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1.  The BCL11A Transcription Factor Directly Activates RAG Gene Expression and V(D)J Recombination 
Molecular and Cellular Biology  2013;33(9):1768-1781.
Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11alox/lox deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.
doi:10.1128/MCB.00987-12
PMCID: PMC3624176  PMID: 23438597
2.  Developmental and species-divergent globin switching are driven by BCL11A 
Nature  2009;460(7259):1093-1097.
The contribution of changes in cis-regulatory elements or trans-acting factors to interspecies differences in gene expression is not well understood. The mammalian β-globin loci have served as a paradigm for gene regulation during development. Transgenic mice harboring the human β-globin locus, consisting of the linked embryonic (ε), fetal (γ) and adult (β) genes, have been used as a model system to study the temporal switch from fetal to adult hemoglobin, as occurs in humans. We show that the human γ-globin genes in these mice behave as murine embryonic globin genes, revealing a limitation of the model and demonstrating that critical differences in the trans-acting milieu have arisen during mammalian evolution. We show that the expression of BCL11A, a repressor of human γ-globin expression identified through genome-wide association studies, differs between mouse and human. Developmental silencing of the mouse embryonic globin and human γ-globin genes fails to occur in mice in the absence of BCL11A. Thus, BCL11A is a critical mediator of species-divergent globin switching. By comparing the ontogeny of β-globin gene regulation in mice and humans, we have shown that alterations in expression of a trans-acting factor constitute a critical driver of gene expression changes during evolution.
doi:10.1038/nature08243
PMCID: PMC3749913  PMID: 19657335
3.  Correction of Sickle Cell Disease in Adult Mice by Interference with Fetal Hemoglobin Silencing 
Science (New York, N.Y.)  2011;334(6058):993-996.
Persistence of human fetal hemoglobin (HbF, α2γ2) in adults lessens the severity of sickle cell disease (SCD) and the β-thalassemias. Here, we show that the repressor BCL11A is required in vivo for silencing of γ-globin expression in adult animals, yet dispensable for red cell production. BCL11A serves as a barrier to HbF reactivation by known HbF inducing agents. In a proof-of-principle test of BCL11A as a potential therapeutic target, we demonstrate that inactivation of BCL11A in SCD transgenic mice corrects the hematologic and pathologic defects associated with SCD through high-level pancellular HbF induction. Thus, interference with HbF silencing by manipulation of a single target protein is sufficient to reverse SCD.
doi:10.1126/science.1211053
PMCID: PMC3746545  PMID: 21998251
4.  Hsp90 – from signal transduction to cell transformation 
The molecular chaperone, Hsp90, facilitates the maturation and/or activation of over 100 ‘client proteins’ involved in signal transduction and transcriptional regulation. Largely an enigma among the families of heat shock proteins, Hsp90 is central to processes broadly ranging from cell cycle regulation to cellular transformation. Here we review the contemporary body of knowledge regarding the biochemical mechanisms of Hsp90 and update the most current paradigms defining its involvement in both normal and pathological cell physiology.
doi:10.1016/j.bbrc.2007.08.054
PMCID: PMC2528838  PMID: 17826744
Hsp90; heat shock protein; molecular chaperone; ATPase; genetic capacitor
5.  Patterns of Spinal Sensory-Motor Connectivity Prescribed by a Dorsoventral Positional Template 
Cell  2011;147(3):653-665.
Summary
Sensory-motor circuits in the spinal cord are constructed with a fine specificity that coordinates motor behavior, but the mechanisms that direct sensory connections with their motor neuron partners remain unclear. The dorsoventral settling position of motor pools in the spinal cord is known to match the distal-to-proximal position of their muscle targets in the limb, but the significance of invariant motor neuron positioning is unknown. An analysis of sensory-motor connectivity patterns in FoxP1 mutant mice, where motor neuron position has been scrambled, shows that the final pattern of sensory-motor connections is initiated by the projection of sensory axons to discrete dorsoventral domains of the spinal cord without regard for motor neuron subtype, or indeed, the presence of motor neurons. By implication, the clustering and dorsoventral settling position of motor neuron pools serves as a determinant of the pattern of sensory input specificity, and thus motor coordination.
doi:10.1016/j.cell.2011.10.012
PMCID: PMC3238499  PMID: 22036571
6.  Characterization of a new ARID family transcription factor (Brightlike/ARID3C) that co-activates Bright/ARID3A-mediated immunoglobulin gene transcription 
Molecular immunology  2011;49(1-2):260-272.
Two members, Bright/ARID3A and Bdp/ARID3B, of the ARID (AT-Rich Interaction Domain) transcription family are distinguished by their ability to specifically bind to DNA and to self-associate via a second domain, REKLES. Bright and Bdp positively regulate immunoglobulin heavy chain gene (IgH) transcription by binding to AT-rich motifs within Matrix Associating Regions (MARs) residing within a subset of VH promoters and the Eµ intronic enhancer. In addition, REKLES provides Bright nuclear export function, and a small pool of Bright is directed to plasma membrane sub-domains/lipid rafts where it associates with and modulates signaling of the B cell antigen receptor (BCR). Here, we characterize a third, highly conserved, physically condensed ARID3 locus, Brightlike/ARID3C. Brightlike encodes two alternatively spliced, SUMO-I-modified isoforms that include or exclude (Δ6) the REKLES-encoding exon 6. Brightlike transcripts and proteins are expressed preferentially within B lineage lymphocytes and coordinate with highest Bright expression--in activated follicular B cells. Brightlike, but not BrightlikeΔ6, undergoes nuclear-cytoplasmic shuttling with a fraction localizing within lipid rafts following BCR stimulation. Brightlike, but not BrightlikeΔ6, associates with Bright in solution, at common DNA binding sites in vitro, and is enriched at Bright binding sites in chromatin. Although possessing little transactivation capacity of its own, Brightlike significantly co-activates Bright-dependent IgH transcription with maximal activity mediated by the unsumoylated form. In sum, this report introduces Brightlike as an additional functional member of the family of ARID proteins, which should be considered in regulatory circuits, previously ascribed to be mediated by Bright.
doi:10.1016/j.molimm.2011.08.025
PMCID: PMC3205283  PMID: 21955986
7.  The L2a element is a mouse CD8 silencer that interacts with MAR-binding proteins SATB1 and CDP 
Molecular immunology  2010;48(1-3):153-163.
Previous transgenic-reporter and targeted-deletion studies indicate that the subset-specific expression of CD8αβ heterodimers is controlled by multiple enhancer activities, since no silencer elements had been found within the locus. We have identified such a silencer as L2a, a previously characterized ~220 bp nuclear matrix associating region (MAR) located ~4.5 kb upstream of CD8α. L2a transgenes driven by the E8I enhancer showed no reporter expression in thymic subsets or T cells in splenic, inguinal and mesenteric lymph node peripheral T cells. Deletion of L2a resulted in significant reporter de-repression, even in the CD4+CD8+ double positive (DP) thymocyte population. L2a contains binding sites for two MAR-interacting proteins, SATB1 and CDP. We found that that binding of these factors was markedly influenced by the content and spacing of L2a sub-motifs (L and S) and that SATB1 binds preferentially to the L motif both in vitro and in vivo. A small fraction of the transgenic CD8+ single positive (SP) thymocytes and peripheral CD8+ T cells bypassed L2a-silencing to give rise to variegated expression of the transgenic reporter. Crossing the L2a-containing transgene onto a SATB1 knockdown background enhanced variegated expression, suggesting that SATB1 is critical in overcoming L2a-silenced transcription.
doi:10.1016/j.molimm.2010.08.014
PMCID: PMC2996921  PMID: 20884053
CD8; transcriptional silencing; transgenic mice; SATB1
8.  The ARID Family Transcription Factor Bright Is Required for both Hematopoietic Stem Cell and B Lineage Development▿  
Molecular and Cellular Biology  2011;31(5):1041-1053.
Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice, its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that >99% of Bright−/− embryos die at midgestation from failed hematopoiesis. Bright−/− embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright−/− mice is markedly reduced. Rare survivors of lethality, which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b, suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody, B-1 responses to phosphocholine, and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation.
doi:10.1128/MCB.01448-10
PMCID: PMC3067827  PMID: 21199920
9.  Loss of Bright/ARID3a Function Promotes Developmental Plasticity 
Stem cells (Dayton, Ohio)  2010;28(9):1560-1567.
Bright (B cell regulator of immunoglobulin heavy chain transcription)/ARID3a, an A+T-rich interaction domain protein, was originally discovered in B lymphocyte lineage cells. However, expression patterns and high lethality levels in knockout mice suggested it had additional functions. Three independent lines of evidence show that functional inhibition of Bright results in increased developmental plasticity. Bright-deficient cells from two mouse models expressed a number of pluripotency-associated gene products, expanded indefinitely and spontaneously differentiated into cells of multiple lineages. Furthermore, direct knockdown of human Bright resulted in colonies capable of expressing multiple lineage markers. These data suggest that repression of this single molecule confers adult somatic cells with new developmental options.
doi:10.1002/stem.491
PMCID: PMC2977942  PMID: 20680960
ARID3a; multipotency
10.  Cardiac Deletion of Smyd2 Is Dispensable for Mouse Heart Development 
PLoS ONE  2010;5(3):e9748.
Chromatin modifying enzymes play a critical role in cardiac differentiation. Previously, it has been shown that the targeted deletion of the histone methyltransferase, Smyd1, the founding member of the SET and MYND domain containing (Smyd) family, interferes with cardiomyocyte maturation and proper formation of the right heart ventricle. The highly related paralogue, Smyd2 is a histone 3 lysine 4- and lysine 36-specific methyltransferase expressed in heart and brain. Here, we report that Smyd2 is differentially expressed during cardiac development with highest expression in the neonatal heart. To elucidate the functional role of Smyd2 in the heart, we generated conditional knockout (cKO) mice harboring a cardiomyocyte-specific deletion of Smyd2 and performed histological, functional and molecular analyses. Unexpectedly, cardiac deletion of Smyd2 was dispensable for proper morphological and functional development of the murine heart and had no effect on global histone 3 lysine 4 or 36 methylation. However, we provide evidence for a potential role of Smyd2 in the transcriptional regulation of genes associated with translation and reveal that Smyd2, similar to Smyd3, interacts with RNA Polymerase II as well as to the RNA helicase, HELZ.
doi:10.1371/journal.pone.0009748
PMCID: PMC2840034  PMID: 20305823
11.  Phosphorylation by SR kinases regulates the binding of PTB-associated splicing factor (PSF) to the pre-mRNA polypyrimidine tract 
FEBS letters  2006;581(2):223-232.
PSF (PTB-associated splicing factor) is a multi-functional protein that participates in transcription and RNA processing. While phosphorylation of PSF has been shown to be important for some functions, the sites and the kinases involved are not well understood. Although PSF does not contain a typical RS domain, we report here that PSF is phosphorylated in vivo to generate an epitope(s) that can be recognized by a monoclonal antibody specific for phosphorylated RS motifs within SR proteins. PSF can be phosphorylated by human and yeast SR kinases in vivo and in vitro at two isolated RS motifs within its N terminus. A functional consequence of SR phosphorylation of PSF is to inhibit its binding to the 3’ polypyrimidine tract of pre-mRNA. These results indicate that PSF is a substrate of SR kinases whose phosphorylation regulates its RNA binding capacity and ultimate biological function.
doi:10.1016/j.febslet.2006.12.015
PMCID: PMC1847627  PMID: 17188683
12.  Open access and beyond 
Molecular Cancer  2006;5:35.
Uncensored exchange of scientific results hastens progress. Open Access does not stop at the removal of price and permission barriers; still, censorship and reading disabilities, to name a few, hamper access to information. Here, we invite the scientific community and the public to discuss new methods to distribute, store and manage literature in order to achieve unfettered access to literature.
doi:10.1186/1476-4598-5-35
PMCID: PMC1569437  PMID: 16956402
13.  A Regulated Nucleocytoplasmic Shuttle Contributes to Bright's Function as a Transcriptional Activator of Immunoglobulin Genes 
Molecular and Cellular Biology  2006;26(6):2187-2201.
Bright/ARID3a has been implicated in mitogen- and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G1/S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.
doi:10.1128/MCB.26.6.2187-2201.2006
PMCID: PMC1430300  PMID: 16507996
14.  Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex 
Molecular Cancer  2006;5:26.
Background
Disrupting the balance of histone lysine methylation alters the expression of genes involved in tumorigenesis including proto-oncogenes and cell cycle regulators. Methylation of lysine residues is commonly catalyzed by a family of proteins that contain the SET domain. Here, we report the identification and characterization of the SET domain-containing protein, Smyd2.
Results
Smyd2 mRNA is most highly expressed in heart and brain tissue, as demonstrated by northern analysis and in situ hybridization. Over-expressed Smyd2 localizes to the cytoplasm and the nucleus in 293T cells. Although accumulating evidence suggests that methylation of histone 3, lysine 36 (H3K36) is associated with actively transcribed genes, we show that the SET domain of Smyd2 mediates H3K36 dimethylation and that Smyd2 represses transcription from an SV40-luciferase reporter. Smyd2 associates specifically with the Sin3A histone deacetylase complex, which was recently linked to H3K36 methylation within the coding regions of active genes in yeast. Finally, we report that exogenous expression of Smyd2 suppresses cell proliferation.
Conclusion
We propose that Sin3A-mediated deacetylation within the coding regions of active genes is directly linked to the histone methyltransferase activity of Smyd2. Moreover, Smyd2 appears to restrain cell proliferation, likely through direct modulation of chromatin structure.
doi:10.1186/1476-4598-5-26
PMCID: PMC1524980  PMID: 16805913
15.  Towards Open Access 
Molecular Cancer  2005;4:20.
doi:10.1186/1476-4598-4-20
PMCID: PMC1180850  PMID: 15932651
16.  PSF Is a Novel Corepressor That Mediates Its Effect through Sin3A and the DNA Binding Domain of Nuclear Hormone Receptors 
Molecular and Cellular Biology  2001;21(7):2298-2311.
Members of the type II nuclear hormone receptor subfamily (e.g., thyroid hormone receptors [TRs], retinoic acid receptors, retinoid X receptors [RXRs], vitamin D receptor, and the peroxisome proliferator-activated receptors) bind to their response sequences with or without ligand. In the absence of ligand, these DNA-bound receptors mediate different degrees of repression or silencing of gene expression which is thought to result from the association of their ligand binding domains (LBDs) with corepressors. Two related corepressors, N-CoR and SMRT, interact to various degrees with the LBDs of these type II receptors in the absence of their cognate ligands. N-CoR and SMRT have been proposed to act by recruiting class I histone deacetylases (HDAC I) through an association with Sin3, although they have also been shown to recruit class II HDACs through a Sin3-independent mechanism. In this study, we used a biochemical approach to identify novel nuclear factors that interact with unliganded full-length TR and RXR. We found that the DNA binding domains (DBDs) of TR and RXR associate with two proteins which we identified as PSF (polypyrimidine tract-binding protein-associated splicing factor) and NonO/p54nrb. Our studies indicate that PSF is a novel repressor which interacts with Sin3A and mediates silencing through the recruitment of HDACs to the receptor DBD. In vivo studies with TR showed that although N-CoR fully dissociates in the presence of ligand, the levels of TR-bound PSF and Sin3A appear to remain unchanged, indicating that Sin3A can be recruited to the receptor independent of N-CoR or SMRT. RXR was not detected to bind N-CoR although it bound PSF and Sin3A as effectively as TR, and this association with RXR did not change with ligand. Our studies point to a novel PSF/Sin3-mediated pathway for nuclear hormone receptors, and possibly other transcription factors, which may fine-tune the transcriptional response as well as play an important role in mediating the repressive effects of those type II receptors which only weakly interact with N-CoR and SMRT.
doi:10.1128/MCB.21.7.2298-2311.2001
PMCID: PMC86864  PMID: 11259580
17.  Cux/CDP Homeoprotein Is a Component of NF-μNR and Represses the Immunoglobulin Heavy Chain Intronic Enhancer by Antagonizing the Bright Transcription Activator 
Molecular and Cellular Biology  1999;19(1):284-295.
Nuclear matrix attachment regions (MARs) flanking the immunoglobulin heavy chain intronic enhancer (Eμ) are the targets of the negative regulator, NF-μNR, found in non-B and early pre-B cells. Expression library screening with NF-μNR binding sites yielded a cDNA clone encoding an alternatively spliced form of the Cux/CDP homeodomain protein. Cux/CDP fulfills criteria required for NF-μNR identity. It is expressed in non-B and early pre-B cells but not mature B cells. It binds to NF-μNR binding sites within Eμ with appropriate differential affinities. Antiserum specific for Cux/CDP recognizes a polypeptide of the predicted size in affinity-purified NF-μNR preparations and binds NF-μNR complexed with DNA. Cotransfection with Cux/CDP represses the activity of Eμ via the MAR sequences in both B and non-B cells. Cux/CDP antagonizes the effects of the Bright transcription activator at both the DNA binding and functional levels. We propose that Cux/CDP regulates cell-type-restricted, differentiation stage-specific Eμ enhancer activity by interfering with the function of nuclear matrix-bound transcription activators.
PMCID: PMC83886  PMID: 9858552
18.  Gene Expression Profile and Functionality of ESC-Derived Lin-ckit+Sca-1+ Cells Are Distinct from Lin-ckit+Sca-1+ Cells Isolated from Fetal Liver or Bone Marrow 
PLoS ONE  2012;7(12):e51944.
In vitro bioreactor-based cultures are being extensively investigated for large-scale production of differentiated cells from embryonic stem cells (ESCs). However, it is unclear whether in vitro ESC-derived progenitors have similar gene expression profiles and functionalities as their in vivo counterparts. This is crucial in establishing the validity of ESC-derived cells as replacements for adult-isolated cells for clinical therapies. In this study, we compared the gene expression profiles of Lin-ckit+Sca-1+ (LKS) cells generated in vitro from mouse ESCs using either static or bioreactor-based cultures, with that of native LKS cells isolated from mouse fetal liver (FL) or bone marrow (BM). We found that in vitro-generated LKS cells were more similar to FL- than to BM LKS cells in gene expression. Further, when compared to cells derived from bioreactor cultures, static culture-derived LKS cells showed fewer differentially expressed genes relative to both in vivo LKS populations. Overall, the expression of hematopoietic genes was lower in ESC-derived LKS cells compared to cells from BM and FL, while the levels of non-hematopoietic genes were up-regulated. In order to determine if these molecular profiles correlated with functionality, we evaluated ESC-derived LKS cells for in vitro hematopoietic-differentiation and colony formation (CFU assay). Although static culture-generated cells failed to form any colonies, they did differentiate into CD11c+ and B220+ cells indicating some hematopoietic potential. In contrast, bioreactor-derived LKS cells, when differentiated under the same conditions failed to produce any B220+ or CD11c+ cells and did not form colonies, indicating that these cells are not hematopoietic progenitors. We conclude that in vitro culture conditions significantly affect the transcriptome and functionality of ESC-derived LKS cells and although in vitro differentiated LKS cells were lineage negative and expressed both ckit and Sca-1, these cells, especially those obtained from dynamic cultures, are significantly different from native cells of the same phenotype.
doi:10.1371/journal.pone.0051944
PMCID: PMC3531429  PMID: 23300581
19.  Functional studies of BCL11A: characterization of the conserved BCL11A-XL splice variant and its interaction with BCL6 in nuclear paraspeckles of germinal center B cells 
Molecular Cancer  2006;5:18.
Background
Chromosomal aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and its deficiency in mice leads to a profound block in B-cell development.
Results
Alternative pre-mRNA splicing of BCL11A produces multiple isoforms sharing a common N-terminus. The most abundant isoform we have identified in human lymphoid samples is BCL11A-XL, the longest transcript produced at this locus, and here we report the conservation of this major isoform and its functional characterization. We show that BCL11A-XL is a DNA-sequence-specific transcriptional repressor that associates with itself and with other BCL11A isoforms, as well as with the BCL6 proto-oncogene. Western blot data for BCL11A-XL expression coupled with data previously published for BCL6 indicates that these genes are expressed abundantly in germinal-center-derived B cells but that expression is extinguished upon terminal differentiation to the plasma cell stage. Although BCL11A-XL/BCL6 interaction can modulate BCL6 DNA binding in vitro, their heteromeric association does not alter the homomeric transcriptional properties of either on model reporter activity. BCL11A-XL partitions into the nuclear matrix and colocalizes with BCL6 in nuclear paraspeckles.
Conclusion
We propose that the conserved N-terminus of BCL11A defines a superfamily of C2HC zinc-finger transcription factors involved in hematopoietic malignancies.
doi:10.1186/1476-4598-5-18
PMCID: PMC1526750  PMID: 16704730

Results 1-19 (19)