Reprogramming human somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variations (CNVs)1-4. To explore this issue, we performed a whole-genome and transcriptome analysis of 20 human iPSC lines derived from primary skin fibroblasts of 7 individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two CNVs not apparent in the fibroblasts from which the iPSC was derived. Using qPCR, PCR, and digital droplet PCR (ddPCR), we show that at least 50% of those CNVs are present as low frequency somatic genomic variants in parental fibroblasts (i.e. the fibroblasts from which each corresponding hiPSC line is derived) and are manifested in iPSC colonies due to the colonies’ clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSC, since most of line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.
The interplay between polycomb and trithorax complexes has been implicated in embryonic stem cell (ESC) self-renewal and differentiation. It has been shown recently that WRD5 and Dpy-30, specific components of the SET1/MLL protein complexes, play important roles during ESC self-renewal and differentiation of neural lineages. However, not much is known about how and where specific trithorax complexes are targeted to genes involved in self-renewal or lineage-specification. Here, we report that the recruitment of the hSET1A histone H3K4 methyltransferase (HMT) complex by transcription factor USF1 is required for mesoderm specification and lineage differentiation. In undifferentiated ESCs, USF1 maintains hematopoietic stem/progenitor cell (HS/PC) associated bivalent chromatin domains and differentiation potential. Furthermore, USF1 directed recruitment of the hSET1A complex to the HoxB4 promoter governs the transcriptional activation of HoxB4 gene and regulates the formation of early hematopoietic cell populations. Disruption of USF or hSET1A function by overexpression of a dominant-negative AUSF1 mutant or by RNA-interference-mediated knockdown, respectively, led to reduced expression of mesoderm markers and inhibition of lineage differentiation. We show that USF1 and hSET1A together regulate H3K4me3 modifications and transcription preinitiation complex assembly at the hematopoietic-associated HoxB4 gene during differentiation. Finally, ectopic expression of USF1 in ESCs promotes mesoderm differentiation and enforces the endothelial-to-hematopoietic transition by inducing hematopoietic-associated transcription factors, HoxB4 and TAL1. Taken together, our findings reveal that the guided-recruitment of the hSET1A histone methyltransferase complex and its H3K4 methyltransferase activity by transcription regulator USF1 safeguards hematopoietic transcription programs and enhances mesoderm/hematopoietic differentiation.
Embryonic stem cells (ESCs) are capable of differentiating into any type of cell or tissue of the body. It is important to understand how developmental genes are controlled during differentiation of ESCs into specific cell types. The hSET1/MLL histone modifying protein complexes add methyl groups to lysine 4 on the N-terminal tails of the DNA associated protein histone H3 and activate gene expression. Although the hSET1/MLL enzymatic complexes play a role in activating genes involved in ESC growth and differentiation, how and where these activities are targeted to remains unclear. In this report, we demonstrate that DNA binding factor USF1 interacts with and brings the hSET1A enzymatic complex to its target gene, HoxB4, during blood cell specification and differentiation. Consistent with the function of HoxB4 in early blood cell formation, we found that the inactivation of USF1 or hSET1A activities leads to a block in the differentiation of blood cells and causes reductions in methylation levels of H3K4 and expression of HoxB4, without impairing the self-renewal capability of ESCs. Taken together, our findings reveal that the collaboration between the hSET1A enzymatic complex and DNA binding regulator USF1 activates developmental genes that control cellular differentiation programs during development.
somatic mosaicism; genomic rearrangement; CNV; aCGH; cancer genomics
To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.
ENCODE Project; mouse genome; DNaseI hypersensitive sites; histone modifications; transcriptome; transcription factor binding sites; comparative genomics; ChIP-seq; RNA-seq
Studies in the area of human brain development are critical as research on neurological and psychiatric disorders has advanced, revealing the origins of pathophysiology to be in the earliest developmental stages. Only with a more precise understanding of the genes and environments that influence the brain in these early stages can we address questions about the pathology, diagnosis, prevention and treatment of neuropsychiatric disorders of developmental origin, like autism, schizophrenia, and Tourette syndrome. A new approach for studying early developmental events is the use of induced pluripotent stem cells (iPSCs). These are cells with wide potential, similar to that of embryonic stem cells, derived from mature somatic cells. We review the protocols used to create iPSCs, including the most efficient and reliable reprogramming strategies available to date for generating iPSCs. In addition, we discuss how this new tool can be applied to neuropsychiatric research. The use of iPSCs can advance our understanding of how genes and gene products are dynamically involved in the formation of unique features of the human brain, and how aberrant genetic variation may interfere with its typical formation. The iPSC technology, if properly applied, can also address basic questions about neural differentiation such as how stem cells can be guided into general and specific neurodevelopmental pathways. Current work in neuropsychiatry with iPSCs derived from patients has focused on disorders with specific genetics deficits and those with less-defined origins; it has revealed previously unknown aspects of pathology and potential pharmacological interventions. These exciting advances based on the use of iPSCs hold promise for improving early diagnosis and, possibly, treatment of psychiatric disorders.
The study of the developing brain has begun to shed light on the underpinnings of both early and adult onset neuropsychiatric disorders. Neuroimaging of the human brain across developmental time points and the use of model animal systems have combined to reveal brain systems and gene products that may play a role in autism spectrum disorders, attention deficit hyperactivity disorder, obsessive compulsive disorder and many other neurodevelopmental conditions. However, precisely how genes may function in human brain development and how they interact with each other leading to psychiatric disorders is unknown. Because of an increasing understanding of neural stem cells and how the nervous system subsequently develops from these cells, we have now the ability to study disorders of the nervous system in a new way—by rewinding and reviewing the development of human neural cells. Induced pluripotent stem cells (iPSCs), developed from mature somatic cells, have allowed the development of specific cells in patients to be observed in real-time. Moreover, they have allowed some neuronal-specific abnormalities to be corrected with pharmacological intervention in tissue culture. These exciting advances based on the use of iPSCs hold great promise for understanding, diagnosing and, possibly, treating psychiatric disorders. Specifically, examination of iPSCs from typically developing individuals will reveal how basic cellular processes and genetic differences contribute to individually unique nervous systems. Moreover, by comparing iPSCs from typically developing individuals and patients, differences at stem cell stages, through neural differentiation, and into the development of functional neurons may be identified that will reveal opportunities for intervention. The application of such techniques to early onset neuropsychiatric disorders is still on the horizon but has become a reality of current research efforts as a consequence of the revelations of many years of basic developmental neurobiological science.
A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34− cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34− cells, using RNA–Seq. We subsequently identified the target genes bound by TCF7, using ChIP–Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34− state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems.
The hematopoietic system has provided a leading model for stem cell studies, and there is great interest in elucidating the mechanisms that control the decision of HSC self-renewal and differentiation. This switch is important for understanding hematopoietic diseases and manipulating HSCs for therapeutic purposes. However, because HSCs are currently unable to proliferate extensively in vitro, this severely limits the types of biochemical analyses that can be performed; and, consequently, the mechanisms that control the decision between early-stage HSC self-renewal and differentiation remain unclear. Murine bone marrow derived EML multipotential hematopoietic precursor cells are ideal for studying the switch. EML cells can grow in large culture and give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34− cells in a cell autonomous fashion. Using RNA–Sequencing and ChIP–Sequencing, we identified and validated the HMG box protein TCF7 as a regulator in this switch and find that it operates in the absence of canonical Wnt signaling. Together with RUNX1, TCF7 regulates a network of transcription factors that characterize the CD34+ cell state. This work serves as a model for studying mechanisms of autonomous and balanced cell fate choice and is ultimately valuable for manipulating HSCs.
PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages.
Cellular identities are established by master regulatory transcription factors that promote cell type–specific gene expression. In some instances such factors also inhibit differentiation of alternative, closely related lineages. PU.1 is an Ets family transcription factor that is required for myeloid and B cell development. PU.1 is also expressed in red blood cell progenitors where it blocks erythroid terminal differentiation. One mechanism used by PU.1 to block red blood cell differentiation is by binding to and inhibiting the erythroid master regulator GATA-1. Here, we describe another mechanism utilized by PU.1 in erythroid progenitors. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) combined with gene expression profiling of erythroid progenitors, we show that PU.1 controls a large gene network in immature erythroid cells, including genes in pathways involved in regulating growth, survival, and differentiation of these cells. We also find that PU.1 controls many of the same genes and pathways in other blood cells. The results indicate that, in addition to activating lineage-specific genes, master regulatory transcription factors, like PU.1, also control numerous, widely expressed genes in multiple cell lineages.
The aim of the present study has been to establish serum free culture conditions for the ex vivo expansion and differentiation of human CD34+ cells into erythroid lineage and to study the chromatin structure, gene expression and transcription factor recruitment at the α–globin locus in the developing erythron.
A basal IMDM cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for the expansion and differentiation of the CD34+ cells. Expression patterns of the alpha and beta like genes at various stages of erythropoiesis was studied by Reverse transcriptase (RT)-qPCR analysis, profile of key erythroid transcription factors was investigated by western blotting, and the chromatin structure and transcription factor recruitment at the alpha globin locus was investigated by ChIP-qPCR analysis.
Human CD34+ cells in the serum free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the α-globin locus during erythroid differentiation of CD34+ cells. NF-E2 was present at upstream activator sites even before addition of erythropoietin (Epo), while bound GATA-1 was only detectable after Epo treatment. After seven days of erythropoietin treatment, H3K4Me2 modification uniformly increases throughout the α–globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2 and GATA-1 were restricted to certain HS sites of the enhancer and theta gene, and were conspicuously low at the α-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the α-globin locus as CD34+ cells differentiated into erythroid pathway.
Our results indicate that remodeling of the upstream elements may be the primary event in activation of α–globin gene expression. Activation of α-globin genes upon Epo treatment involves initial binding of Pol II, down-regulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1.
Differences in gene expression may play a major role in speciation and phenotypic diversity. We examined genome-wide differences in transcription factor (TF) binding in several humans and a single chimpanzee using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). The binding sites of RNA Polymerase II (PolII) and a key regulator of immune responses, NFκB (p65), were mapped in ten lymphoblastoid cell lines and 25% and 7.5% of the respective binding regions were found to differ between individuals. Binding differences were frequently associated with SNPs and genomic structural variants (SVs) and were often correlated with differences in gene expression, suggesting functional consequences of binding variation. Furthermore, comparing PolII binding between human and chimpanzee suggests extensive divergence in TF binding. Our results indicate that many differences in individuals and species occur at the level of TF binding and provide insight into the genetic events responsible for these differences.
Erythrocyte membrane protein genes serve as excellent models of complex gene locus structure and function, but their study has been complicated by both their large size and their complexity. To begin to understand the intricate interplay of transcription, dynamic chromatin architecture, transcription factor binding, and genomic organization in regulation of erythrocyte membrane protein genes, we performed chromatin immunoprecipitation (ChIP) coupled with microarray analysis and ChIP coupled with massively parallel DNA sequencing in both erythroid and nonerythroid cells. Unexpectedly, most regions of GATA-1 and NF-E2 binding were remote from gene promoters and transcriptional start sites, located primarily in introns. Cooccupancy with FOG-1, SCL, and MTA-2 was found at all regions of GATA-1 binding, with cooccupancy of SCL and MTA-2 also found at regions of NF-E2 binding. Cooccupancy of GATA-1 and NF-E2 was found frequently. A common signature of histone H3 trimethylation at lysine 4, GATA-1, NF-E2, FOG-1, SCL, and MTA-2 binding and consensus GATA-1-E-box binding motifs located 34 to 90 bp away from NF-E2 binding motifs was found frequently in erythroid cell-expressed genes. These results provide insights into our understanding of membrane protein gene regulation in erythropoiesis and the regulation of complex genetic loci in erythroid and nonerythroid cells and identify numerous candidate regions for mutations associated with membrane-linked hemolytic anemia.
mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgene-mediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3+ CD8+, CD3+ CD4+, and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3+CD8+ lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD/SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.
Following recent technological advances there has been an increasing interest in genome structural variation, in particular copy-number variants (CNVs) – large-scale duplications and deletions – in the human genome. Although not immediately evident, CNV surveys make a conceptual connection between the fields of population genetics and protein families, in particular with regard to the stability and expandability of families. The mechanisms giving rise to CNVs can be considered as fundamental processes underlying gene duplication and loss; duplicated genes being the results of “successful” copies, fixed and maintained in the population. Conversely, many “unsuccessful” duplicates remain in the genome as pseudogenes. Here, we survey studies on CNVs, highlighting issues related to protein families. In particular, CNVs tend to affect specific gene functional categories, such as those associated with environmental response, and are depleted in genes related to basic cellular processes. Furthermore, CNVs occur more often at the periphery of the protein interaction network. Thereby, functional categories associated with successful duplicates and unsuccessful duplicates are clearly distinguishable. These trends are likely reflective of CNV formation biases and natural selection, both of which differentially influence distinct protein families.
Structural variation of the genome involves kilobase- to megabase-sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements. We introduce high-throughput and massive paired-end mapping (PEM), a large-scale genome-sequencing method to identify structural variants (SVs) ~3 kilobases (kb) or larger that combines the rescue and capture of paired ends of 3-kb fragments, massive 454 sequencing, and a computational approach to map DNA reads onto a reference genome. PEM was used to map SVs in an African and in a putatively European individual and identified shared and divergent SVs relative to the reference genome. Overall, we fine-mapped more than 1000 SVs and documented that the number of SVs among humans is much larger than initially hypothesized; many of the SVs potentially affect gene function. The breakpoint junction sequences of more than 200 SVs were determined with a novel pooling strategy and computational analysis. Our analysis provided insights into the mechanisms of SV formation in humans.
The sterol regulatory element-binding protein (SREBP) family member SREBP1 is a critical transcriptional regulator of cholesterol and fatty acid metabolism and has been implicated in insulin resistance, diabetes, and other diet-related diseases. We globally identified the promoters occupied by SREBP1 and its binding partners NFY and SP1 in a human hepatocyte cell line using chromatin immunoprecipitation combined with genome tiling arrays (ChIP-chip). We find that SREBP1 occupies the promoters of 1,141 target genes involved in diverse biological pathways, including novel targets with roles in lipid metabolism and insulin signaling. We also identify a conserved SREBP1 DNA-binding motif in SREBP1 target promoters, and we demonstrate that many SREBP1 target genes are transcriptionally activated by treatment with insulin and glucose using gene expression microarrays. Finally, we show that SREBP1 cooperates extensively with NFY and SP1 throughout the genome and that unique combinations of these factors target distinct functional pathways. Our results provide insight into the regulatory circuitry in which SREBP1 and its network partners coordinate a complex transcriptional response in the liver with cues from the diet.
Transcription factors (TFs) are DNA-binding proteins that regulate the transcription of their target genes. TFs typically bind in proximity to the transcription start sites of their target genes in a region called the promoter. SREBP1 is a TF that increases the transcription of numerous genes involved in cholesterol and fat metabolism and has been linked to diet-related diseases such as insulin resistance and type 2 diabetes. Using microarray technology, we identified all of the promoters in the human genome that are bound by SREBP1 and two associated TFs called NFY and SP1 in a human liver cell line. Our findings greatly expand the number of genes and biological pathways that may be regulated by SREBP1 and reveal that different combinations of SREBP1 and its partners preferentially target genes involved in different pathways. Thus, in contrast to traditional studies that focus on individual genes, we have used a genomics approach to provide a novel global view of the regulatory circuitry in which SREBP1 and its partners coordinate a transcriptional response in the liver with cues from the diet.
Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.
Traditional microarrays use probes complementary to known genes to quantitate the differential gene expression between two or more conditions. Genomic tiling microarray experiments differ in that probes that span a genomic region at regular intervals are used to detect the presence or absence of transcription. This difference means the same sets of biases and the methods for addressing them are unlikely to be relevant to both types of experiment. We introduce the informatics challenges arising in the analysis of tiling microarray experiments as open problems to the scientific community and present initial approaches for the analysis of this nascent technology.
RACE sequencing of ENCODE regions shows that much of the human genome is represented in poly(A)+ RNA.
Recent studies of the mammalian transcriptome have revealed a large number of additional transcribed regions and extraordinary complexity in transcript diversity. However, there is still much uncertainty regarding precisely what portion of the genome is transcribed, the exact structures of these novel transcripts, and the levels of the transcripts produced.
We have interrogated the transcribed loci in 420 selected ENCyclopedia Of DNA Elements (ENCODE) regions using rapid amplification of cDNA ends (RACE) sequencing. We analyzed annotated known gene regions, but primarily we focused on novel transcriptionally active regions (TARs), which were previously identified by high-density oligonucleotide tiling arrays and on random regions that were not believed to be transcribed. We found RACE sequencing to be very sensitive and were able to detect low levels of transcripts in specific cell types that were not detectable by microarrays. We also observed many instances of sense-antisense transcripts; further analysis suggests that many of the antisense transcripts (but not all) may be artifacts generated from the reverse transcription reaction. Our results show that the majority of the novel TARs analyzed (60%) are connected to other novel TARs or known exons. Of previously unannotated random regions, 17% were shown to produce overlapping transcripts. Furthermore, it is estimated that 9% of the novel transcripts encode proteins.
We conclude that RACE sequencing is an efficient, sensitive, and highly accurate method for characterization of the transcriptome of specific cell/tissue types. Using this method, it appears that much of the genome is represented in polyA+ RNA. Moreover, a fraction of the novel RNAs can encode protein and are likely to be functional.
The FAT10 gene encodes a diubiquitin-like protein containing two tandem head-to-tail ubiquitin-like domains. There is a high degree of similarity between murine and human FAT10 sequences at both the mRNA and protein levels. In various cell lines, FAT10 expression was shown to be induced by gamma interferon or by tumor necrosis factor alpha. In addition, FAT10 expression was found to be up-regulated in some Epstein-Barr virus-infected B-cell lines, in activated dendritic cells, and in several epithelial tumors. However, forced expression of FAT10 in cultured cells was also found to produce apoptotic cell death. Overall, these findings suggest that FAT10 may modulate cellular growth or cellular viability. Here we describe the steps to generate, by genetic targeting, a FAT10 gene knockout mouse model. The FAT10 knockout homozygous mice are viable and fertile. No gross lesions or obvious histological differences were found in these mutated mice. Examination of lymphocyte populations from spleen, thymus, and bone marrow did not reveal any abnormalities. However, flow cytometry analysis demonstrated that the lymphocytes of FAT10 knockout mice were, on average, more prone to spontaneous apoptotic death. Physiologically, these mice demonstrated a high level of sensitivity toward endotoxin challenge. These findings indicate that FAT10 may function as a survival factor.
Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of γH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the lesions. Furthermore, WRN RNAi-induced DNA damage was suppressed by overexpression of the telomere-binding protein TRF2. These conditions, however, did not prevent the DNA damage response in BLM-ablated cells, suggesting a distinct role for WRN in DNA homeostasis in vivo. Thus, manifestations of Werner syndrome may reflect an impaired ability of slowly dividing cells to limit oxidative DNA damage.
The cyclic AMP-responsive element-binding protein (CREB) is an important transcription factor that can be activated by hormonal stimulation and regulates neuronal function and development. An unbiased, global analysis of where CREB binds has not been performed. We have mapped for the first time the binding distribution of CREB along an entire human chromosome. Chromatin immunoprecipitation of CREB-associated DNA and subsequent hybridization of the associated DNA to a genomic DNA microarray containing all of the nonrepetitive DNA of human chromosome 22 revealed 215 binding sites corresponding to 192 different loci and 100 annotated potential gene targets. We found binding near or within many genes involved in signal transduction and neuronal function. We also found that only a small fraction of CREB binding sites lay near well-defined 5′ ends of genes; the majority of sites were found elsewhere, including introns and unannotated regions. Several of the latter lay near novel unannotated transcriptionally active regions. Few CREB targets were found near full-length cyclic AMP response element sites; the majority contained shorter versions or close matches to this sequence. Several of the CREB targets were altered in their expression by treatment with forskolin; interestingly, both induced and repressed genes were found. Our results provide novel molecular insights into how CREB mediates its functions in humans.
We studied the genetic and engraftment phenotype of highly purified murine hematopoietic stem cells (lineage negative, rhodamine-low, Hoechst-low) through cytokine-stimulated cell cycle. Cells were cultured in interleukin (IL)-3, IL-6, IL-11, and steel factor for 0 to 48 h and tested for engraftment capacity in a lethally irradiated murine competitive transplant model. Engraftment showed major fluctuations with nadirs at 36 and 48 h of culture and recovery during the next G1. Gene expression of quiescent (0 h) or cycling (48 h) stem cells was compared with lineage positive cells by 3′ end PCR differential display analysis. Individual PCR bands were quantified using a 0 to 9 scale and results were visually compared using color-coded matrices. We defined a set of 637 transcripts expressed in stem cells and not expressed in lineage positive cells. Gene expression analyzed at 0 and 48 h showed a major shift from “stem cell genes” being highly expressed at 0 h and turned off at 48 h, while “cell division” genes were turned on at 48 h. These observations suggest stem cell gene expression shifts through cell cycle in relation to cell cycle related alterations of stem cell phenotype. The engraftment defect is related to a major phenotypic change of the stem cell.
stem; cell; gene; expression; cycle