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1.  Translating Dosage Compensation to Trisomy 21 
Nature  2013;500(7462):10.1038/nature12394.
Down syndrome (DS) is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21 (Chr21). We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST. Using genome editing with zinc finger nucleases, we targeted a large, inducible XIST transgene into the Chr21 DYRK1A locus, in DS pluripotent stem cells. XIST RNA coats Chr21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a “Chr21 Barr Body.” This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Remarkably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one Chr21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of “chromosome therapy”.
doi:10.1038/nature12394
PMCID: PMC3848249  PMID: 23863942
2.  XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells 
Background
The establishment of facultative heterochromatin by X-chromosome inactivation requires the long non-coding RNA XIST/Xist. However, the molecular mechanism by which the RNA achieves chromosome-wide gene silencing remains unknown. Mouse Xist has been shown to have redundant domains for cis-localization, and requires a series of well-conserved tandem ‘A’ repeats for silencing. We previously described a human inducible XIST transgene that is capable of cis-localization and suppressing a downstream reporter gene in somatic cells, and have now leveraged these cells to dissect the sequences critical for XIST-dependent gene silencing in humans.
Results
We demonstrated that expression of the inducible full-length XIST cDNA was able to suppress expression of two nearby reporter genes as well as endogenous genes up to 3 MB from the integration site. An inducible construct containing the repeat A region of XIST alone could silence the flanking reporter genes but not the more distal endogenous genes. Reporter gene silencing could also be accomplished by a synthetic construct consisting of nine copies of a consensus repeat A sequence, consistent with previous studies in mice. Progressively shorter constructs showed a linear relationship between the repeat number and the silencing capacity of the RNA. Constructs containing only two repeat A units were still able to partially silence the reporter genes and could thus be used for site-directed mutagenesis to demonstrate that sequences within the two palindromic cores of the repeat are essential for silencing, and that it is likely the first palindrome sequence folds to form a hairpin, consistent with compensatory mutations observed in eutherian sequences.
Conclusions
Silencing of adjacent reporter genes can be effected by as little as 94 bp of XIST, including two ‘monomers’ of the A repeat. This region includes a pair of essential palindromic sequences that are evolutionarily well-conserved and the first of these is likely to form an intra-repeat hairpin structure. Additional sequences are required for the spread of silencing to endogenous genes on the chromosome.
doi:10.1186/1756-8935-6-23
PMCID: PMC3734131  PMID: 23915978
X-chromosome inactivation; XIST; Long non-coding RNA; Eutherian dosage compensation; Gene silencing
3.  The Effect of Changes in Stimulus Level on Electrically Evoked Cortical Auditory Potentials 
Ear and hearing  2009;30(3):320-329.
Objectives
The purpose of this study was to determine whether the electrically evoked acoustic change complex (EACC) could be used to assess sensitivity to changes in stimulus level in cochlear implant (CI) recipients and to investigate the relationship between EACC amplitude and rate of growth of the N1-P2 onset response with increases in stimulus level.
Design
Twelve postlingually deafened adults using Nucleus CI24 CIs participated in this study. Nucleus Implant Communicator (NIC) routines were used to bypass the speech processor and to control the stimulation of the implant directly. The stimulus consisted of an 800 msec burst of a 1000 pps biphasic pulse train. A change in the stimulus level was introduced 400 msec after stimulus onset. Band-pass filtering (1 to 100 Hz) was used to minimize stimulus artifact. Four to six recordings of 50 sweeps were obtained for each condition, and averaged responses were analyzed in the time domain using standard peak picking procedures.
Results
Cortical auditory change potentials were recorded from CI users in response to both increases and decreases in stimulation level. The amplitude of the EACC was found to be dependent on the magnitude of the stimulus change. Increases in stimulus level elicited more robust EACC responses than decreases in stimulus level. Also, EACC amplitudes were significantly correlated with the slope of the growth of the onset response.
Conclusions
This work describes the effect of change in stimulus level on electrically evoked auditory change potentials in CI users. The amplitude of the EACC was found to be related both to the magnitude of the stimulus change introduced and to the rate of growth of the N1-P2 onset response. To the extent that the EACC reflects processing of stimulus change, it could potentially be a valuable tool for assessing neural processing of the kinds of stimulation patterns produced by a CI. Further studies are needed, however, to determine the relationships between the EACC and psychophysical measures of intensity discrimination in CI recipients.
doi:10.1097/AUD.0b013e31819c42b7
PMCID: PMC2896017  PMID: 19322089
4.  Effects of Long-Term Use of a Cochlear Implant on the Electrically Evoked Compound Action Potential 
Background
Since the early 1990s, it has been possible to measure electrically evoked compound action potentials (ECAPs) from Nucleus cochlear implant users. Recording the ECAP does not require active participation by the subject, and the recordings are not adversely affected by attention or sleep, making this response an ideal tool for monitoring long-term changes. Previous research from our laboratory (Hughes et al, 2001) has shown that ECAP thresholds and slope of the ECAP growth functions are relatively stable over time. However, this conclusion was based on results obtained from a fairly limited number of study participants, each of whom used the Nucleus CI24M cochlear implant and were followed for less than two years.
Purpose
To evaluate the effect of long-term use of a cochlear implant on ECAP thresholds and slope of the ECAP input/output function for both pediatric and adult cochlear implant recipients.
Research Design
A longitudinal study that describes how ECAP thresholds and growth functions change over a period of 96 mo following initial activation. Changes over time in ECAP threshold and slope of the ECAP growth function were analyzed, and effects of the subject’s age, type of CI (cochlear implant), and stimulating electrode are included in the analysis.
Study Sample
134 Nucleus CI users participated in this study. All were profoundly deaf. This subject pool included 84 individuals (40 adults and 44 children) who used the Nucleus CI24M cochlear implant and 50 individuals (21 adults and 29 children) who used the Nucleus CI24R cochlear implant.
Data Collection and Analysis
Electrodes 5, 10, 15, and 20 were stimulated, and ECAP growth functions were measured for each subject at regular intervals following the initial activation of the device.
Results
Small increases in mean ECAP thresholds were observed for both pediatric and adult CI users between an “early” visit that occurred within 3–6 mo following hookup and a “late” visit that occurred 4.8–6 yr later. For adults, the average increase in ECAP threshold was 3.94 CL (clinical programming units for Nucleus CIs). For children, the average increase was 4.16 CL. These differences, while small, were statistically significant. Slope of the ECAP growth functions measured over the same time interval did not change significantly. On average, pediatric CI users had ECAP thresholds that were 4–5 CL units higher than the adult CI recipients. The most striking outcome from this study, however, was the finding that when compared with postlingually deafened adults, pediatric CI users had ECAP growth functions that were substantially steeper. The differences between the results obtained from children and those obtained from adults were statistically significant and largely independent of device type or stimulating electrode.
Conclusion
Results from this study show ECAP thresholds and growth functions to change very little over a 5–6 yr observation interval suggesting that long-term use of a CI is not likely to have a significant negative impact on the response of the peripheral auditory system. Pediatric CI users were shown to have, on average, higher ECAP thresholds and steeper ECAP growth functions than postlingually deafened adult CI users. This finding suggests potential differences between the two patient populations either in terms of the current fields within the cochlea or the effective distance between the stimulating electrode and the stimulable neural tissue.
PMCID: PMC2881552  PMID: 20085195
Auditory evoked potential; cochlear implant; compound action potential; electrical stimulation; neural response telemetry
5.  A skewed view of X chromosome inactivation 
X chromosome inactivation involves a random choice to silence either X chromosome early in mammalian female development. Once silenced the inactive X is stably inherited through subsequent somatic cell divisions, and thus, females are generally mosaics, having a mixture of cells with one or the other parental X active. While in most females the number of cells with either X being active is roughly equal, skewing of X chromosome inactivation is observed in a percentage of women. In this issue of the JCI, Bolduc and colleagues address whether skewing of X chromosome inactivation in humans is influenced by an X-linked locus that can alter this initial random inactivation (see the related article beginning on page 333). Their data indicate that most of the skewing observed in humans results from secondary events rather than being due to an inherited tendency to inactivate a particular X chromosome.
doi:10.1172/JCI34470
PMCID: PMC2147673  PMID: 18097476
6.  Additional annotation enhances potential for biologically-relevant analysis of the Illumina Infinium HumanMethylation450 BeadChip array 
Background
Measurement of genome-wide DNA methylation (DNAm) has become an important avenue for investigating potential physiologically-relevant epigenetic changes. Illumina Infinium (Illumina, San Diego, CA, USA) is a commercially available microarray suite used to measure DNAm at many sites throughout the genome. However, it has been suggested that a subset of array probes may give misleading results due to issues related to probe design. To facilitate biologically significant data interpretation, we set out to enhance probe annotation of the newest Infinium array, the HumanMethylation450 BeadChip (450 k), with >485,000 probes covering 99% of Reference Sequence (RefSeq) genes (National Center for Biotechnology Information (NCBI), Bethesda, MD, USA). Annotation that was added or expanded on includes: 1) documented SNPs in the probe target, 2) probe binding specificity, 3) CpG classification of target sites and 4) gene feature classification of target sites.
Results
Probes with documented SNPs at the target CpG (4.3% of probes) were associated with increased within-tissue variation in DNAm. An example of a probe with a SNP at the target CpG demonstrated how sample genotype can confound the measurement of DNAm. Additionally, 8.6% of probes mapped to multiple locations in silico. Measurements from these non-specific probes likely represent a combination of DNAm from multiple genomic sites. The expanded biological annotation demonstrated that based on DNAm, grouping probes by an alternative high-density and intermediate-density CpG island classification provided a distinctive pattern of DNAm. Finally, variable enrichment for differentially methylated probes was noted across CpG classes and gene feature groups, dependant on the tissues that were compared.
Conclusion
DNAm arrays offer a high-throughput approach for which careful consideration of probe content should be utilized to better understand the biological processes affected. Probes containing SNPs and non-specific probes may affect the assessment of DNAm using the 450 k array. Additionally, probe classification by CpG enrichment classes and to a lesser extent gene feature groups resulted in distinct patterns of DNAm. Thus, we recommend that compromised probes be removed from analyses and that the genomic context of DNAm is considered in studies deciphering the biological meaning of Illumina 450 k array data.
doi:10.1186/1756-8935-6-4
PMCID: PMC3740789  PMID: 23452981
Infinium HumanMethylation450 BeadChip array; DNA methylation; non-specific probes; Polymorphic probes; CpG islands; Annotation; CpG enrichment; Tissue-specific DNA methylation; Repetitive elements; 450 k
7.  Preliminary results of the relationship between the binaural interaction component of the electrically evoked auditory brainstem response and interaural pitch comparisons in bilateral cochlear implant recipients 
Ear and hearing  2012;33(1):57-68.
Objectives
The purpose of this study was to investigate the relationship between electrophysiologic measures of the binaural interaction component (BIC) of the electrically evoked auditory brainstem response (EABR) and psychophysical measures of interaural pitch comparisons in Nucleus bilateral cochlear implant users.
Design
Data were collected for ten postlingually deafened adult cochlear implant users. Each subject conducted an interaural pitch-comparison task using a biphasic pulse train with a pulse rate of 1000 pulses per second (pps) at high stimulation levels. Stimuli were presented in a two-interval, two-alternative forced-choice procedure with roving current variations. A subgroup of four subjects repeated the task at low stimulation levels. BICs were measured using loudness balanced, biphasic current pulses presented at a rate of 19.9 pps for each subject by pairing the electrode 12 (out of 22 intracochlear electrodes) in the right ear with each of 11 electrodes spaced across the electrode array in the left ear. The BIC was measured at high stimulation levels in ten subjects and at low stimulation levels in seven subjects. Because of differences in stimulation rate used in BIC measures and interaural pitch comparisons, the actual stimulation levels were different in these two measures. The relationship between BIC responses and results of interaural pitch comparisons was evaluated for each of the individual subjects as well as at the group level. Evaluation was carried out separately for results obtained at high and low stimulation levels.
Results
There was no significant correlation between results of BIC measures and interaural pitch comparisons on either the individual or group levels. Lower stimulation level did not improve the relationship between these two measures.
Conclusions
No significant correlations between psychophysical measures of interaural pitch comparisons and electrophysiologic measures of the BIC of the EABR were found. The lack of correlation may be attributed to methods used to quantify the data, small number of subjects retested at low stimulation levels, as well as central processing components involved in the interaural pitch-comparison task.
doi:10.1097/AUD.0b013e31822519ef
PMCID: PMC3193893  PMID: 21730858
auditory evoked potential; bilateral cochlear implantation; auditory brain stem response; electrical stimulation
8.  Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing 
Genetics  2012;192(4):1281-1293.
Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.
doi:10.1534/genetics.112.143743
PMCID: PMC3512139  PMID: 23023002
X chromosome inactivation (XCI); DNA methylation; human transgenes; targeted transgenesis; Xist deletion
9.  The Relationship between Electrically Evoked Compound Action Potential and Speech Perception : A Study in Cochlear Implant Users with Short Electrode Array 
Objectives
To determine the extent to which electrically evoked compound action potential (ECAP) measurements were related with speech perception performance in implant users with a short electrode array and to investigate the relationship between ECAP measures and performance according to specific devices.
Design
Prospective study.
Setting
Tertiary referral center.
Patients
Seventeen Hybrid cochlear implant users were tested in this study. Subjects were divided into 2 groups: 1) 8 using the Nucleus Hybrid M, 2) 9 using the Nucleus Hybrid RE. In addition, 21 Nucleus Freedom long electrode implant (CI24RE) users were also tested to compare with the results of the old device (CI24M).
Main Outcome measures
ECAP growth functions were recorded using either an interphase gap (IPG) of 8 or 45 us. We then calculated 1) the slope of the growth function, and 2) changes in sensitivity with IPG. For each subject, these measures were compared with performance on tests of word recognition.
Results
The changes in sensitivity using two IPGs showed no correlation with results of word recognition test in Hybrid cochlear implant users. In contrast, relatively strong correlations have been found between the slope of ECAP growth functions and performance on word recognition test. Additionally, when we separate the results of Hybrid M and RE, the slopes of ECAP growth functions from only Hybrid RE CI recipients were significantly correlated with speech performance. The slopes of ECAP growth function in CI24RE users with long electrode were also significantly correlated with performance. However, comparing between two independent correlations in RE devices, correlation was higher in Hybrid RE group.
Conclusion
The results presented in this paper support the view that slope of the ECAP growth can show significant correlation to performance with a cochlear implant. Further, these results suggest that the strength of the correlation may be related to the specific device. These results suggest that ECAP measures may be useful in developing a test to predict outcomes with the implant.
doi:10.1097/MAO.0b013e3181ec1d92
PMCID: PMC2933654  PMID: 20634770
electrically evoked compound action potential; cochlear implant; Hybrid electrodes
10.  Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation 
Human Genetics  2011;130(2):187-201.
X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E−16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset of genes predicted different XCI statuses in different tissues. Using previously published expression data the effect of transcription on gene-body methylation was investigated and while X-linked introns of highly expressed genes were more methylated than the introns of lowly expressed genes, exonic methylation did not differ based on expression level. We conclude that the XCI status predicted using methylation of X-linked promoters with CpG islands was usually the same as determined by expression analysis and that 12% of X-linked genes examined show tissue-specific XCI whereby a gene has a different XCI status in at least one of the four tissues examined.
Electronic supplementary material
The online version of this article (doi:10.1007/s00439-011-1007-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00439-011-1007-8
PMCID: PMC3132437  PMID: 21597963
11.  The functional role of long non-coding RNA in human carcinomas 
Molecular Cancer  2011;10:38.
Long non-coding RNAs (lncRNAs) are emerging as new players in the cancer paradigm demonstrating potential roles in both oncogenic and tumor suppressive pathways. These novel genes are frequently aberrantly expressed in a variety of human cancers, however the biological functions of the vast majority remain unknown. Recently, evidence has begun to accumulate describing the molecular mechanisms by which these RNA species function, providing insight into the functional roles they may play in tumorigenesis. In this review, we highlight the emerging functional role of lncRNAs in human cancer.
doi:10.1186/1476-4598-10-38
PMCID: PMC3098824  PMID: 21489289
12.  Methylated DNA Immunoprecipitation 
The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.
doi:10.3791/935
PMCID: PMC2763296  PMID: 19241501
13.  Comparison of Electrically Evoked Compound Action Potential Thresholds and Loudness Estimates for the Stimuli Used to Program the Advanced Bionics Cochlear Implant 
Background
In the mid-1990s, Cochlear Corporation introduced a cochlear implant (CI) to the market that was equipped with hardware that made it possible to record electrically evoked compound action potentials (ECAPs) from CI users of all ages. Over the course of the next decade, many studies were published that compared ECAP thresholds with levels used to program the speech processor of the Nucleus CI. In 2001 Advanced Bionics Corporation introduced the Clarion CII cochlear implant (the Clarion CII internal device is also known as the CII Bionic Ear). This cochlear implant was also equipped with a system that allowed measurement of the ECAP. While a great deal is known about how ECAP thresholds compare with the levels used to program the speech processor of the Nucleus CI, relatively few studies have reported comparisons between ECAP thresholds and the levels used to program the speech processor of the Advanced Bionics CI.
Purpose
To explore the relationship between ECAP thresholds and behavioral measures of perceptual dynamic range for the range of stimuli commonly used to program the speech processor of the Advanced Bionics CI.
Research Design
This prospective and experimental study uses correlational and descriptive statistics to define the relationship between ECAP thresholds and perceptual dynamic range measures.
Study Sample
Twelve postlingually deafened adults participated in this study. All were experienced users of the Advanced Bionics CI system.
Data Collection and Analysis
ECAP thresholds were recorded using the commercially available SoundWave software. Perceptual measures of threshold (T-level), most comfortable level (M-level), and maximum comfortable level (C-level) were obtained using both “tone bursts” and “speech bursts.” The relationship between these perceptual and electrophysiological variables was defined using paired t-tests as well as correlation and linear regression.
Results
ECAP thresholds were significantly correlated with the perceptual dynamic range measures studied; however, correlations were not strong. Analysis of the individual data revealed considerable discrepancy between the contour of ECAP threshold versus electrode function and the behavioral loudness estimates used for programming.
Conclusion
ECAP thresholds recorded from Advanced Bionics cochlear implant users always indicated levels where the programming stimulus was audible for the listener. However, the correlation between ECAP thresholds and M-levels (the primary metric used to program the speech processor of the Advanced Bionics CI), while statistically significant, was quite modest. If programming levels are to be determined on the basis of ECAP thresholds, care should be taken to ensure that stimulation is not uncomfortably loud, particularly on the basal electrodes in the array.
PMCID: PMC2875933  PMID: 20085196
Auditory evoked potential; cochlear implant; compound action potential; electrical stimulation; neural response imaging
14.  Active Chromatin Marks Are Retained on X Chromosomes Lacking Gene or Repeat Silencing Despite XIST/Xist Expression in Somatic Cell Hybrids 
PLoS ONE  2010;5(5):e10787.
Background
X-chromosome inactivation occurs early in mammalian development and results in the inactive X chromosome acquiring numerous hallmarks of heterochromatin. While XIST is a key player in the inactivation process, the method of action of this ncRNA is yet to be determined.
Methodology/Principal Findings
To assess which features of heterochromatin may be directly recruited by the expression and localization of the XIST RNA we have analyzed a mouse/human somatic cell hybrid in which expression of human and mouse XIST/Xist has been induced from the active X by demethylation. Such hybrids had previously been demonstrated to disconnect XIST/Xist expression from gene silencing and we confirm maintenance of X-linked gene expression, even close to the Xist locus, despite the localized expression of mouse Xist.
Conclusions/Significance
Loss of the active chromatin marks H3 acetylation and H3 lysine 4 methylation was not observed upon XIST/Xist expression, nor was there a gain of DNA methylation; thus these marks of facultative heterochromatin are not solely dependent upon Xist expression. Cot-1 holes, regions of depleted RNA hybridization with a Cot-1 probe, were observed upon Xist expression; however, these were at reduced frequency and intensity in these somatic cells. Domains of human Cot-1 transcription were observed corresponding to the human chromosomes in the somatic cell hybrids. The Cot-1 domain of the X was not reduced with the expression of XIST, which fails to localize to the human X chromosome in a mouse somatic cell background. The human inactive X in a mouse/human hybrid cell also shows delocalized XIST expression and an ongoing Cot-1 domain, despite X-linked gene silencing. These results are consistent with recent reports separating Cot-1 silencing from genic silencing, but also demonstrate repetitive element expression from an otherwise silent X chromosome in these hybrid cells.
doi:10.1371/journal.pone.0010787
PMCID: PMC2875404  PMID: 20520737
15.  Identification of regulatory elements flanking human XIST reveals species differences 
BMC Molecular Biology  2010;11:20.
Background
The transcriptional silencing of one X chromosome in eutherians requires transcription of the long non-coding RNA gene, XIST. Many regulatory elements have been identified downstream of the mouse Xist gene, including the antisense Tsix gene. However, these elements do not show sequence conservation with humans, and the human TSIX gene shows critical differences from the mouse. Thus we have undertaken an unbiased identification of regulatory elements both downstream and upstream of the human XIST gene using DNase I hypersensitivity mapping.
Results
Downstream of XIST a single DNase I hypersensitive site was identified in a mouse undifferentiated ES cell line containing an integration of the human XIC region. This site was not observed in somatic cells. Upstream of XIST, the distance to the flanking JPX gene is expanded in humans relative to mice, and we observe a hypersensitive site 65 kb upstream of XIST, in addition to hypersensitive sites near the XIST promoter. This -65 region has bi-directional promoter activity and shows sequence conservation in non-rodent eutheria.
Conclusions
The lack of regulatory elements corresponding to human TSIX lends further support to the argument that TSIX is not a regulator of XIST in humans. The upstream hypersensitive sites we identify show sequence conservation with other eutheria, but not with mice. Therefore the regulation of XIST seems to be different between mice and man, and regulatory sequences upstream of XIST may be important regulators of XIST in non-rodent eutheria instead of Tsix which is critical for Xist regulation in rodents.
doi:10.1186/1471-2199-11-20
PMCID: PMC2841178  PMID: 20211024
16.  The Electrically Evoked Auditory Change Complex: Preliminary Results from Nucleus Cochlear Implant Users 
Ear and hearing  2008;29(5):704-717.
Objectives
The purpose of this study was to determine if changes in the position of the stimulating electrode in the cochlea could be used to elicit the electrically evoked auditory change complex (EACC) from Nucleus cochlear implant users.
Design
Nine postlingually deafened adults participated in this study. Each study participant had been using his or her Nucleus CI24 cochlear implant for at least 3 months prior to testing. The speech processor was bypassed and the output of the implanted receiver/stimulator was controlled directly. The stimulus was a 600 ms burst of a biphasic pulse train (1000 pps). In control conditions, the stimulating electrode was held constant and stimulation continued throughout the 600 ms recording interval. In experimental conditions, the EACC was elicited by introducing a change in the stimulating electrode 300 ms after the onset of the pulse train. The EACC was recorded using surface electrodes. Three recordings of 100 sweeps each were obtained for each stimulus condition. Bandpass filtering (1 – 100 Hz) was used to minimize contamination of the recordings by stimulus artifact. Averaged responses were then smoothed using a 40 ms wide boxcar filter and standard peak picking procedures were used to analyze these responses in the time domain.
Results
In each case, a clear onset response (P1-N1-P2) was recorded. In the experimental conditions, a second evoked potential, the EACC, was also recorded following the change in stimulating electrode. This second response had general morphologic characteristics that were very similar to those of the onset response. Increasing the separation between the two stimulating electrodes in the experimental conditions resulted in a general trend toward increased EACC amplitudes.
Conclusions
This report describes results of a set of experiments in which the speech processor of the cochlear implant was bypassed and the EACC was recorded in response to a change in stimulating electrode position. EACC amplitude was shown to increase as the separation between the two stimulating electrodes increased. While preliminary in nature, these results demonstrate the feasibility of recording the EACC in response to changes in stimulating electrode position from individual cochlear implant users.
doi:10.1097/AUD.0b013e31817a98af
PMCID: PMC2767236  PMID: 18596644
auditory evoked potential; cochlear implant; cortical evoked potential; electrical stimulation
17.  Inactive X chromosome-specific reduction in placental DNA methylation 
Human Molecular Genetics  2009;18(19):3544-3552.
Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the placenta has been reported to demonstrate a global decrease in methylation as well as decreased methylation of X-linked promoters. Methylation is one of many features that differentiate the active and inactive X, and it is well established that CpG island promoters on the inactive X are hypermethylated. We now report a detailed analysis of methylation at different regions across the X in male and female placenta and blood. A significant (P < 0.001) placental hypomethylation of LINE1 elements was observed in both males and females. Relative to blood placental promoter hypomethylation was only observed for X-linked, not autosomal promoters, and was significant for females (P < 0.0001) not males (P = 0.9266). In blood, X-linked CpG island promoters were shown to have moderate female methylation (66% across 70 assays) and low (23%) methylation in males. A similar methylation pattern in blood was observed for ∼20% of non-island promoters as well as 50% of the intergenic or intragenic CpG islands, the latter is likely due to the presence of unannotated promoters. Both intragenic and intergenic regions showed similarly high methylation levels in male and female blood (68 and 66%) while placental methylation of these regions was lower, particularly in females. Thus placental hypomethylation relative to blood is observed globally at repetitive elements as well as across the X. The decrease in X-linked placental methylation is consistently greater in females than males and implicates an inactive X specific loss of methylation in the placenta.
doi:10.1093/hmg/ddp299
PMCID: PMC2742397  PMID: 19586922
19.  BCoR-L1 variation and breast cancer 
Introduction
BRCA1 is involved in numerous essential processes in the cell, and the effects of BRCA1 dysfunction in breast cancer carcinogenesis are well described. Many of the breast cancer susceptibility genes such as BRCA2, p53, ATM, CHEK2, and BRIP1 encode proteins that interact with BRCA1. BCL6 corepressor-like 1 (BCoR-L1) is a newly described BRCA1-interacting protein that displays high homology to several proteins known to be involved in the fundamental processes of DNA damage repair and transcription regulation. BCoR-L1 has been shown to play a role in transcription corepression, and expression of the X-linked BCoR-L1 gene has been reported to be dysregulated in breast cancer subjects. BCoR-L1 is located on the X chromosome and is subject to X inactivation.
Methods
We performed mutation analysis of 38 BRCA1/2 mutation-negative breast cancer families with male breast cancer, prostate cancer, and/or haplotype sharing around BCoR-L1 to determine whether there is a role for BCoR-L1 as a high-risk breast cancer predisposition gene. In addition, we conducted quantitative real-time PCR (qRT-PCR) on lymphoblastoid cell lines (LCLs) from the index cases from these families and a number of cancer cell lines to assess the role of BCoR-L1 dysregulation in cancer and cancer families.
Results
Very little variation was detected in the coding region, and qRT-PCR analysis revealed that BCoR-L1 expression is highly variable in cancer-free subjects, high-risk breast cancer patients, and cancer cell lines. We also report the investigation of a new expression control, DIDO1 (death inducer-obliterator 1), that is superior to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and UBC (ubiquitin C) for analysis of expression in LCLs.
Conclusion
Our results suggest that BCoR-L1 expression does not play a large role in predisposition to familial breast cancer.
doi:10.1186/bcr1759
PMCID: PMC2206730  PMID: 17697391
20.  Epigenetic predisposition to expression of TIMP1 from the human inactive X chromosome 
BMC Genetics  2005;6:48.
Background
X inactivation in mammals results in the transcriptional silencing of an X chromosome in females, and this inactive X acquires many of the epigenetic features of silent chromatin. However, not all genes on the inactive X are silenced, and we have examined the TIMP1 gene, which has variable inactivation amongst females. This has allowed us to examine the features permitting expression from the otherwise silent X by comparing inactive X chromosomes with and without TIMP1 expression.
Results
Expression was generally correlated with euchromatic chromatin features, including DNA hypomethylation, nuclease sensitivity, acetylation of histone H3 and H4 and hypomethylation of H3 at lysines 9 and 27. Demethylation of the TIMP1 gene by 5-azacytidine was able to induce expression from the inactive X chromosome in somatic cell hybrids, and this expression was also accompanied by features of active chromatin. Acetylated histone H3 continued to be observed even when expression was lost in cells that naturally expressed TIMP1; while acetylation was lost upon TIMP1 silencing in cells where expression from the inactive X had been induced by demethylation. Thus ongoing acetylation of inactive X chromosomes does not seem to be simply a 'memory' of expression.
Conclusion
We propose that acetylation of H3 is an epigenetic mark that predisposes to TIMP1 expression from the inactive X chromosome in some females.
doi:10.1186/1471-2156-6-48
PMCID: PMC1262707  PMID: 16194278
21.  Stabilization and Localization of Xist RNA are Controlled by Separate Mechanisms and are Not Sufficient for X Inactivation  
The Journal of Cell Biology  1998;142(1):13-23.
These studies address whether XIST RNA is properly localized to the X chromosome in somatic cells where human XIST expression is reactivated, but fails to result in X inactivation (Tinker, A.V., and C.J. Brown. 1998. Nucl. Acids Res. 26:2935–2940). Despite a nuclear RNA accumulation of normal abundance and stability, XIST RNA does not localize in reactivants or in naturally inactive human X chromosomes in mouse/ human hybrid cells. The XIST transcripts are fully stabilized despite their inability to localize, and hence XIST RNA localization can be uncoupled from stabilization, indicating that these are separate steps controlled by distinct mechanisms. Mouse Xist RNA tightly localized to an active X chromosome, demonstrating for the first time that the active X chromosome in somatic cells is competent to associate with Xist RNA. These results imply that species-specific factors, present even in mature, somatic cells that do not normally express Xist, are necessary for localization. When Xist RNA is properly localized to an active mouse X chromosome, X inactivation does not result. Therefore, there is not a strict correlation between Xist localization and chromatin inactivation. Moreover, expression, stabilization, and localization of Xist RNA are not sufficient for X inactivation. We hypothesize that chromosomal association of XIST RNA may initiate subsequent developmental events required to enact transcriptional silencing.
PMCID: PMC2133021  PMID: 9660859
22.  Human Cancer Long Non-Coding RNA Transcriptomes 
PLoS ONE  2011;6(10):e25915.
Once thought to be a part of the ‘dark matter’ of the genome, long non-coding RNAs (lncRNAs) are emerging as an integral functional component of the mammalian transcriptome. LncRNAs are a novel class of mRNA-like transcripts which, despite no known protein-coding potential, demonstrate a wide range of structural and functional roles in cellular biology. However, the magnitude of the contribution of lncRNA expression to normal human tissues and cancers has not been investigated in a comprehensive manner. In this study, we compiled 272 human serial analysis of gene expression (SAGE) libraries to delineate lncRNA transcription patterns across a broad spectrum of normal human tissues and cancers. Using a novel lncRNA discovery pipeline we parsed over 24 million SAGE tags and report lncRNA expression profiles across a panel of 26 different normal human tissues and 19 human cancers. Our findings show extensive, tissue-specific lncRNA expression in normal tissues and highly aberrant lncRNA expression in human cancers. Here, we present a first generation atlas for lncRNA profiling in cancer.
doi:10.1371/journal.pone.0025915
PMCID: PMC3185064  PMID: 21991387

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