NuRD Chromatin Remodeling Complex Research Articles

EPCO-27. CHROMATIN REMODELING REPROGRAMS THE TRANSCRIPTION NETWORK IN GLIOBLASTOMA

Abstract Fundamentally, cancer arises from genetic and epigenetic alterations that interplay to reprogram gene expression networks, leading to unrestrained proliferation. Glioblastoma (GBM) is the most prevalent and aggressive primary brain cancer, with a median survival of approximately one year and a 5-year survival rate of just 5%. The standard care for GBM patients has remained unchanged for decades, underscoring the need for a better understanding of GBM biology to develop more effective therapies. The relatively low genetic mutation burden in GBM highlights the significant role of epigenetic mechanisms in its pathogenesis. Our recent work shows that chromatin remodeling plays pivotal roles in GBM pathogenesis. The BRD8-driven EP400 chromatin remodeling complex maintains GBM by crippling p53-mediated tumor suppression in an unprecedented way through hijacking the histone variant H2AZ at p53 target loci, enforcing a repressive chromatin state that prevents p53-mediated transactivation. Targeting BRD8 in TP53WT GBM, which makes up ~71% of all GBM cases, enhances chromatin accessibility by evicting H2AZ. This restores p53-mediated transactivation of its targets, reestablishes cell cycle arrest, inhibits gliomagenesis, and prolongs survival in xenograft models of TP53WT GBM. Conversely, the NuRD chromatin remodeling complex driven by CHD5 governs the MYC-mediated oncogenic network to prevent gliomagenesis. Mechanistically, CHD5 forms a CHD3-containing but CHD4-independent NuRD complex that directly binds to the MYC promoter and super enhancers. Chd5 loss triggers the remodeling of chromatin, enhances chromatin accessibility at Myc loci, augments Myc expression, significantly impairs NSCs differentiation while promotes proliferation, transformation, and gliomagenesis in vivo. Furthermore, reactivating CHD5 in human GBM xenograft models significantly extends survival. Thus, our findings reveal previously unappreciated epigenetic mechanisms by which GBM cells reprogram both tumor-suppressive and oncogenic transcription networks to facilitate their outgrowth. This sheds new light on our understanding of GBM malignancy and provides promising therapeutic opportunities for treating patients with this devastating malignancy.

Developmental analysis of Spalt function in the Drosophila prothoracic gland.

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope.

Cell-fate conversion of intestinal cells in adult Drosophila midgut by depleting a single transcription factor

The manipulation of cell identity by reprograming holds immense potential in regenerative medicine, but is often limited by the inefficient acquisition of fully functional cells. This problem can potentially be resolved by better understanding the reprogramming process using in vivo genetic models, which are currently scarce. Here we report that both enterocytes (ECs) and enteroendocrine cells (EEs) in adult Drosophila midgut show a surprising degree of cell plasticity. Depleting the transcription factor Tramtrack in the differentiated ECs can initiate Prospero-mediated cell transdifferentiation, leading to EE-like cells. On the other hand, depletion of Prospero in the differentiated EEs can lead to the loss of EE-specific transcription programs and the gain of intestinal progenitor cell identity, allowing cell cycle re-entry or differentiation into ECs. We find that intestinal progenitor cells, ECs, and EEs have a similar chromatin accessibility profile, supporting the concept that cell plasticity is enabled by pre-existing chromatin accessibility with switchable transcription programs. Further genetic analysis with this system reveals that the NuRD chromatin remodeling complex, cell lineage confliction, and age act as barriers to EC-to-EE transdifferentiation. The establishment of this genetically tractable in vivo model should facilitate mechanistic investigation of cell plasticity at the molecular and genetic level.

CHD4 Promotes Tumor Formation, Survival, and Resistance to Genotoxic Therapeutics in TP53 Mutant T-Cell Acute Lymphoblastic Leukemia Cells through the NuRD Chromatin Remodeling Complex

T-Cell acute leukemia(T-ALL) though generally responsive to current chemotherapy remains incurable in over 50% of adult patients. Importantly, the survival for patients with TP53 mutant T-ALL disease is less than 10%. We have previously shown the role of the Methyl Binding Domain protein 2-Nucleosome Remodeling and Deacetylase (MBD2-NURD) chromatin remodeling complex in triple negative breast cancer cell growth and the role of the NuRD component, Chromatin-helicase-binding protein 4(CHD4), in tumor colony formation and genotoxic resistance in acute myeloid leukemia. In the present study, we have examined the role of specific NuRD complexes and CHD4 in T-cell acute leukemia cells with mutant TP53. The prototypical NuRD complex possesses both a histone deacetylase (HDCC) sub-complex and an ATPase chromatin remodeling sub-complex containing CHD4 linked to MBD2 or MBD3 of the HDCC. CHD4 is linked to the MBD2/3 anchors of the NuRD complex, via GATAD2A or GATAD2B. Depletion of CHD4 was found to increase the rate of apoptosis in several TP53 mutant T-ALL cell lines including Jurkat, MOLT4, RPMI and CCRF by up to 5 to 10-fold. Likewise, depletion of both MBD2 and MBD3 also increased apoptosis and sensitization to the standard genotoxic chemotherapeutic agent, cytosine arabinoside (Ara-C) but had only minimal effects in normal human CD34+ hematopoietic progenitor cells. Depletion of GATAD2B and GATAD2A by shRNA also resulted in a significantly increased survival of 60 days compared to 20 days in Scramble shRNA controls, in a xenogeneic mouse model of T-ALL. Depletion of CHD4 resulted in a profound (10 to 100-fold) decrease in colony formation of the TP53 mutant T-ALL cell lines in methylcellulose. RNA seq analyses identified the apoptosis, ferroptosis and mitophagy pathways as potential mechanisms for the striking effect of NURD disruption on colony formation. In summary, CHD4, through the NuRD complex, promotes tumor cell formation, survival and therapeutic resistance of TP53 mutant T-ALL cells. Current efforts are aimed at validating these findings in primary T-ALL cells and structure-function studies to advance development of tool compounds for disrupting the NuRD complex, with the long-term goal of developing improved small molecule therapeutics for treatment resistant acute leukemia.

The chromatin-remodeling complexes B-WICH and NuRD regulate ribosomal transcription in response to glucose.

Regulation of ribosomal transcription is under tight control from environmental stimuli, and this control involves changes in the chromatin structure. The underlying mechanism of how chromatin changes in response to nutrient and energy supply in the cell is still unclear. The chromatin-remodeling complex B-WICH is involved in activating the ribosomal transcription, and we show here that knock down of the B-WICH component WSTF results in cells that do not respond to glucose. The promoter is less accessible, and RNA pol I and its transcription factors SL1/TIF-1B and RRN3/TIF-1A, as well as the proto-oncogene c-MYC and the activating deacetylase SIRT7 do not bind upon glucose stimulation. In contrast, the repressive chromatin state that forms after glucose deprivation is reversible, and RNA pol I factors are recruited. WSTF knock down results in an accumulation of the ATPase CHD4, a component of the NuRD chromatin remodeling complex, which is responsible for establishing a repressive poised state at the promoter. The TTF-1, which binds and affect the binding of the chromatin complexes, is important to control the association of activating chromatin component UBF. We suggest that B-WICH is required to allow for a shift to an active chromatin state upon environmental stimulation, by counteracting the repressive state induced by the NuRD complex.

Akirin Is Required for Muscle Function and Acts Through the TGF-β Sma/Mab Signaling Pathway in Caenorhabditis elegans Development.

Akirin, a conserved metazoan protein, functions in muscle development in flies and mice. However, this was only tested in the rodent and fly model systems. Akirin was shown to act with chromatin remodeling complexes in transcription and was established as a downstream target of the NFκB pathway. Here we show a role for Caenorhabditis elegans Akirin/AKIR-1 in the muscle and body length regulation through a different pathway. Akirin localizes to somatic tissues throughout the body of C. elegans, including muscle nuclei. In agreement with its role in other model systems, Akirin loss of function mutants exhibit defects in muscle development in the embryo, as well as defects in movement and maintenance of muscle integrity in the C. elegans adult. We also have determined that Akirin acts downstream of the TGF-β Sma/Mab signaling pathway in controlling body size. Moreover, we found that the loss of Akirin resulted in an increase in autophagy markers, similar to mutants in the TGF-β Sma/Mab signaling pathway. In contrast to what is known in rodent and fly models, C. elegans Akirin does not act with the SWI/SNF chromatin-remodeling complex, and is instead involved with the NuRD chromatin remodeling complex in both movement and regulation of body size. Our studies define a novel developmental role (body size) and a new pathway (TGF-β Sma/Mab) for Akirin function, and confirmed its evolutionarily conserved function in muscle development in a new organism.

MUC1-C Activates the NuRD Complex to Drive Dedifferentiation of Triple-Negative Breast Cancer Cells.

The NuRD chromatin remodeling and deacetylation complex, which includes MTA1, MBD3, CHD4, and HDAC1 among other components, is of importance for development and cancer progression. The oncogenic mucin 1 (MUC1) C-terminal subunit (MUC1-C) protein activates EZH2 and BMI1 in the epigenetic reprogramming of triple-negative breast cancer (TNBC). However, there is no known link between MUC1-C and chromatin remodeling complexes. Here, we showed that MUC1-C binds directly to the MYC HLH-LZ domain and identified a previously unrecognized MUC1-C→MYC pathway that regulates the NuRD complex. MUC1-C/MYC complexes selectively activated the MTA1 and MBD3 genes and posttranscriptionally induced CHD4 expression in basal- but not luminal-type BC cells. In turn, MUC1-C formed complexes with these NuRD components on the ESR1 promoter. Downregulating MUC1-C decreased MTA1/MBD3/CHD4/HDAC1 occupancy and increased H3K27 acetylation on the ESR1 promoter, with induction of ESR1 expression and downstream estrogen response pathways. Targeting MUC1-C and these NuRD components also induced expression of FOXA1, GATA3, and other markers associated with the luminal phenotype. These findings support a model in which MUC1-C activates the NuRD complex to drive dedifferentiation and reprogramming of TNBC cells. SIGNIFICANCE: MUC1-C directly interacts with MYC to activate the NuRD complex, mediating regulation of the estrogen receptor in triple-negative breast cancer cells.

Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex

Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

Direct interaction between the PRDM3 and PRDM16 tumor suppressors and the NuRD chromatin remodeling complex.

Aberrant isoform expression of chromatin-associated proteins can induce epigenetic programs related to disease. The MDS1 and EVI1 complex locus (MECOM) encodes PRDM3, a protein with an N-terminal PR-SET domain, as well as a shorter isoform, EVI1, lacking the N-terminus containing the PR-SET domain (ΔPR). Imbalanced expression of MECOM isoforms is observed in multiple malignancies, implicating EVI1 as an oncogene, while PRDM3 has been suggested to function as a tumor suppressor through an unknown mechanism. To elucidate functional characteristics of these N-terminal residues, we compared the protein interactomes of the full-length and ΔPR isoforms of PRDM3 and its closely related paralog, PRDM16. Unlike the ΔPR isoforms, both full-length isoforms exhibited a significantly enriched association with components of the NuRD chromatin remodeling complex, especially RBBP4. Typically, RBBP4 facilitates chromatin association of the NuRD complex by binding to histone H3 tails. We show that RBBP4 binds to the N-terminal amino acid residues of PRDM3 and PRDM16, with a dissociation constant of 3.0 μM, as measured by isothermal titration calorimetry. Furthermore, high-resolution X-ray crystal structures of PRDM3 and PRDM16 N-terminal peptides in complex with RBBP4 revealed binding to RBBP4 within the conserved histone H3-binding groove. These data support a mechanism of isoform-specific interaction of PRDM3 and PRDM16 with the NuRD chromatin remodeling complex.

Mbd2-CP2c loop drives adult-type globin gene expression and definitive erythropoiesis.

During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.

A Functional Switch of NuRD Chromatin Remodeling Complex Subunits Regulates Mouse Cortical Development

SummaryHistone modifications and chromatin remodeling represent universal mechanisms by which cells adapt their transcriptional response to rapidly changing environmental conditions. Extensive chromatin remodeling takes place during neuronal development, allowing the transition of pluripotent cells into differentiated neurons. Here, we report that the NuRD complex, which couples ATP-dependent chromatin remodeling with histone deacetylase activity, regulates mouse brain development. Subunit exchange of CHDs, the core ATPase subunits of the NuRD complex, is required for distinct aspects of cortical development. Whereas CHD4 promotes the early proliferation of progenitors, CHD5 facilitates neuronal migration and CHD3 ensures proper layer specification. Inhibition of each CHD leads to defects of neuronal differentiation and migration, which cannot be rescued by expressing heterologous CHDs. Finally, we demonstrate that NuRD complexes containing specific CHDs are recruited to regulatory elements and modulate the expression of genes essential for brain development.

Epigenetic Regulation of Cell Cycle-Promoting Genes By Ikaros and HDAC1 in Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood leukemia. Expression profiling has identified IKZF1 (Ikaros) as a major tumor suppressor in B-ALL and established reduced Ikaros function as a poor prognostic marker for this disease. Ikaros regulates expression of its target genes via chromatin remodeling. In vivo, Ikaros can form a complex with histone deacetylases HDAC1 and/or HDAC2 as well as the NuRD chromatin remodeling complex. The mechanisms by which Ikaros exerts its tumor suppressor function and regulates gene expression in B-ALL are unknown. Here we report the use of chromatin immunoprecipitation coupled with next generation sequencing (ChIP-SEQ) to identify genes that are regulated by Ikaros in vivo and to determine the role of Ikaros in chromatin remodeling in B-ALL.Results reveal that Ikaros binds to the promoter regions of a large number of genes that are critical for cell cycle progression. These include CDC2, CDC16, CDC25A, ANAPC1, and ANAPC7. Overexpression of Ikaros in leukemia cells resulted in transcriptional repression of Ikaros target genes. Results from luciferase reporter assays performed using the respective promoters of Ikaros target genes support a role for Ikaros as a transcriptional repressor of these genes. Downregulation of Ikaros by siRNA resulted in increased expression of Ikaros target genes that control cell cycle progression. These results suggest that Ikaros functions as a negative regulator of cell cycle progression by repressing transcription of cell cycle-promoting genes. Next, we studied how Ikaros binding affects the epigenetic signature at promoters of Ikaros target genes. Global epigenetic mapping showed that Ikaros binding at the promoter region of cell cycle-promoting genes is associated with the formation of one of two types of repressive epigenetic marks – either H3K27me3 or H3K9me3. While these epigenetic marks were mutually exclusive, they were both associated with the loss of H3K9 acetylation and transcriptional repression. Serial qChIP assays spanning promoters of the Ikaros target genes revealed that the presence of H3K27me3 is associated with Ikaros and HDAC1 binding, while the H3K9me3 modification is associated with Ikaros binding and the absence of HDAC1. ChIP-SEQ analysis of HDAC1 global genomic binding demonstrated that over 80% of H3K27me3 modifications at promoter regions are associated with HDAC1 binding at surrounding sites. The treatment of leukemia cells with the histone deacetylase inhibitor – trichostatin (TSA) resulted in a severe reduction of global levels of H3K27me3, as evidenced by Wesern blot. These data suggest that HDAC1 activity in leukemia is essential for the formation of repressive chromatin that is characterized by the presence of H3K27me3.Our data suggest that Ikaros binding at the promoters of its target genes can result in the formation of repressive chromatin by two distinct mechanisms: 1) direct Ikaros binding resulting in increased H3K9me3 or 2) Ikaros recruitment of HDAC1 with increased H3K27me3 modifications. These data suggest distinct mechanisms for the regulation of chromatin remodeling and target gene expression by Ikaros alone, and Ikaros in complex with HDAC1. In conclusion, the presented data suggest that HDAC1 has a key role in regulating cell cycle progression and proliferation in B-ALL. Our results identify novel, Ikaros-mediated mechanisms of epigenetic regulation that contribute to tumor suppression in leukemia. Supported by National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment. DisclosuresNo relevant conflicts of interest to declare.

Promoter Decommissioning by the NuRD Chromatin Remodeling Complex Triggers Synaptic Connectivity in the Mammalian Brain

Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by invivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex invivo. By interfacing genome-wide sequencing of transcripts and ChIP-seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum invivo. Finally, in a targeted invivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally regulated programming mechanism that drives synaptic connectivity in the mammalian brain.

HIV-1 Vpr Induces the Degradation of ZIP and sZIP, Adaptors of the NuRD Chromatin Remodeling Complex, by Hijacking DCAF1/VprBP

The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered.

A role of Zeb2 in the development of terminally differentiated effector T cells (P1434)

Abstract Zeb2 (also known as Zfhx1b and Sip1) is a DNA-binding multi-zinc-finger transcription factor found to play a significant role in the development of cells both early in neuronal differentiation and in cancer metastasis. By binding dual E2 box enhancer elements, Zeb2 acts primarily as a transcriptional repressor and may inhibit E-protein DNA binding, although it is known to act also as a transcriptional activator. Zeb2 also binds activated Smads, the co-repressor CtBP and the NuRD chromatin remodeling complex but not all of its functions may necessarily depend on each of these interactions. While the closely related family member, dEG1/Zfhx1a/Zeb1, is known to plays an early role in lymphocyte development, the function of Sip1 in T lymphocytes remains unknown. We find that Zeb2 is upregulated in activated T cells and that antigen specific T cells show diminished expansion during an immune response. Zeb2 is selectively expressed and necessary for the development of a subset of terminally differentiated CD8+ T cells. We are currently assessing the interplay between Zeb2 and E-proteins in differentiation of naïve CD8+ T cells to cytotoxic effector cells.

Landscape of somatic single-nucleotide and copy-number mutations in uterine serous carcinoma

Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer. We analyzed the mutational landscape of USC by whole-exome sequencing of 57 cancers, most of which were matched to normal DNA from the same patients. The distribution of the number of protein-altering somatic mutations revealed that 52 USC tumors had fewer than 100 (median 36), whereas 5 had more than 3,000 somatic mutations. The mutations in these latter tumors showed hallmarks of defects in DNA mismatch repair. Among the remainder, we found a significantly increased burden of mutation in 14 genes. In addition to well-known cancer genes (i.e., TP53, PIK3CA, PPP2R1A, KRAS, FBXW7), there were frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling complex, and TAF1, an element of the core TFIID transcriptional machinery. Additionally, somatic copy-number variation was found to play an important role in USC, with 13 copy-number gains and 12 copy-number losses that occurred more often than expected by chance. In addition to loss of TP53, we found frequent deletion of a small segment of chromosome 19 containing MBD3, also a member of the NuRD-chromatin-modification complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (an upstream activator of PIK3CA), and CCNE1 (a target of FBXW7-mediated ubiquitination). These findings identify frequent mutation of DNA damage, chromatin remodeling, cell cycle, and cell proliferation pathways in USC and suggest potential targets for treatment of this lethal variant of endometrial cancer.

An intrinsically unstructured domain in MBD2 recruits the histone deacetylase core complex of NuRD and modifies kinetics of DNA binding

MBD2 specifically recognizes methylated DNA and recruits the NuRD chromatin remodeling complex to silence expression of the associated gene. We recently solved the structure of a coiled‐coil interaction between MBD2 and p66α that recruits the Mi2 nucleosome remodeling protein to the complex (Gnanapragasam et al., Proc Natl Acad Sci. 2011, 108:7487). In subsequent work, we have shown that a region between the methyl‐cytosine binding (MBD) and the coiled‐coil domains of MBD2 stably binds the histone deacetylase core complex of NuRD comprised by HDAC1/2, MTA1/2, and RbAp46/48. NMR and CD studies demonstrate that this region is largely unstructured even in the context of full‐length protein and while bound to methylated DNA. Immunoprecipitation studies show that the critical determinant of protein binding resides within two small molecular recognition fragments (MoRFs) that together are sufficient to recruit the core deacetylase complex. Previous analyses showed very rapid association and dissociation rates for the isolated MBD. In contrast, incorporation of the intrinsically unstructured domain reduces the association rate and markedly prolongs dissociation from methylated DNA. Therefore, this intrinsically unstructured domain functions both to recruit a large portion of the NuRD complex and stabilize interaction with methylated DNA. Supported by ACS IRG‐73‐001 (DCW) and NIH R01‐DK029902 (GDG).

Ikaros changes the face of NuRD remodeling

The inclusion of the transcription factor Ikaros in the NuRD chromatin-remodeling complex regulates both the targeting and activity of the NuRD complex in lymphocytes, thereby influencing developmental gene-expression programs.

Abstract 935: Protein phosphatase 1 (PP1) regulates tumor suppressor activity of Ikaros in leukemia cells

Abstract Ikaros encodes a DNA-binding zinc finger protein that acts as a tumor suppressor in human leukemia. The essential components of Ikaros’ activity include DNA binding and transcriptional repression via chromatin remodeling. We have previously reported that Ikaros directly interacts with Protein Phosphatase 1 (PP1) and undergoes dephosphorylation at specific amino acids (Popescu et al. J Biol Chem 2009 284(20):13869). PP1-mediated dephosphorylation of Ikaros regulates its DNA-binding to probes derived from gamma satellites of pericentromeric heterochromatin, and controls subcellular, pericentromeric localization of Ikaros in vivo. We extended our studies to determine how PP1-mediated dephoshorylation of Ikaros controls Ikaros’ function in regulating the expression of its target genes. An Ikaros mutant that is unable to associate with PP1 (IK 465/7A), and consequently hyperphosphorylated was compared to the wild type Ikaros for: 1) DNA-binding affinity toward the promoters of Ikaros’ target genes; 2) interaction with the NuRD chromatin remodeling complex; and 3) ability to repress Ikaros’ target genes using a luciferase reporter assay. Results showed that the lack of dephosphorylation by PP1 and the resulting increase in phosphorylation of Ikaros is responsible for its inability to bind the promoters of several Ikaros target genes. The absence of interaction with PP1 inhibits binding of Ikaros to the chromatin remodeling complex NuRD. In addition, the inability to interact with PP1 phosphatase abolished Ikaros’ repressor function as evidenced by failure of the IK 465/7A mutant to repress transcription of luciferase in the reporter assay. These results established that dephosphorylation of Ikaros by PP1 is essential for its function as a regulator of gene expression. Studies using an Ikaros mutant that is unable to interact with PP1, but that also contains 11 alanine (phosphoresistant) mutations at CK2 phosphorylation sites (IK 465/7A+A11 mutant) established which of these functional changes are the result of CK2-mediated phosphorylation. Results showed that IK 465/7A+A11 is able to bind to the Ikaros consensus biding sites at promoters of the Ikaros target genes, and to associate with the NuRD remodeling complex. Inhibition of Ikaros phosphorylation by CK2 kinase also restored Ikaros’ ability to repress transcription of target genes in the luciferase reporter assay. Thus, the introduction of 11 alanine mutations at CK2 phosphorylation sites restored the DNA-binding ability and chromatin remodeling function, as well as the transcriptional repressor function of the Ikaros mutant despite its inability to interact with PP1. Our results suggest that dephosphorylation of Ikaros by PP1 and its phosphorylation by CK2 kinase are two opposing mechanisms that control Ikaros’ function in the regulation of gene expression in leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 935. doi:10.1158/1538-7445.AM2011-935

Oct1 Is a Switchable, Bipotential Stabilizer of Repressed and Inducible Transcriptional States

Little is known regarding how the Oct1 transcription factor regulates target gene expression. Using murine fibroblasts and two target genes, Polr2a and Ahcy, we show that Oct1 recruits the Jmjd1a/KDM3A lysine demethylase to catalyze the removal of the inhibitory histone H3K9 dimethyl mark and block repression. Using purified murine T cells and the Il2 target locus, and a colon cancer cell line and the Cdx2 target locus, we show that Oct1 recruits the NuRD chromatin-remodeling complex to promote a repressed state, but in a regulated manner can switch to a different capacity and mediate Jmjd1a recruitment to block repression. These findings indicate that Oct1 maintains repression through a mechanism involving NuRD and maintains poised gene expression states through an antirepression mechanism involving Jmjd1a. We propose that, rather than acting as a primary trigger of gene activation or repression, Oct1 is a switchable stabilizer of repressed and inducible states.