Transcriptional Repressor Complex Research Articles

Abstract A22: STAG2, a novel genetic regulator of bladder cancer

Abstract STAG2 is known as a cohesin complex component, and frequent loss-of-function mutations in bladder cancer (BC) suggest its importance in tumorigenesis. While STAG2 functions in chromatid segregation and colocalizes with CTCF to organize chromatin, we provide evidence that STAG2 may have roles independent of this canonical function. Importantly, unique STAG2 binding sites may correspond to tissue-specific enhancers. We hypothesize that STAG2 acts as a transcription factor that influences BC. Whole- and targeted-exome sequencing of muscle-invasive (MIBC) patient tissues (n=119) revealed a STAG2 mutation in over 10%. Tumor immunohistochemistry confirmed loss of STAG2 protein expression. We observed that patients (n=330) with no STAG2 expression had prolonged median overall (34 vs. 24.5 months, p=.049) and progression-free survival (23 vs. 13.5 months, p=.016) compared to STAG2-positive tumors. To evaluate the functional impact of STAG2 status, we stably knocked down STAG2 in T24 cells. We observed decreased invasion in shSTAG2 cells via transwell migration assays and a significant increase in cisplatin sensitivity. To determine STAG2 interactions with chromatin, we conducted ChIP-seq (WT STAG2) and RNA-seq (upon STAG2 knockdown) in T24 cells. We identified STAG2-dependent dysregulation of epithelial-to-mesenchymal transition genes including MMP2, MMP9, SLUG, and SNAIL, consistent with STAG2's role in invasion. Cistrome pipeline integration of ChIP-seq and RNA-seq data suggests a repressive role for STAG2 (p=.000503). Overlap of STAG2 targets from Cistrome with GeneHancer revealed 2,811 enhancers that STAG2 may regulate. Finally, STAG2 rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) identified 154 potential transcription cofactors that immunoprecipitated with STAG2 in the context of chromatin. As expected, we identified known components of the cohesin complex, such as RAD21, SMC1A, and SMC3. However, many of the proteins identified through RIME have not been previously found to interact with STAG2 as cohesins. Interestingly, some of these STAG2-interacting proteins have been implicated in the regulation of gene expression, such as EIF4A1 and NONO, further supporting our hypothesis that STAG2 is part of a transcriptional repression complex that is important for BC. Our ongoing analyses of STAG2 will reveal if STAG2 enhancer binding is independent of its role in chromatin organization. We suggest that STAG2 may bind to specific enhancers and repress transcription in BC. Loss of STAG2 may push disease towards a less invasive phenotype and may also confer sensitivity to cisplatin treatment. These results are consistent with the idea that loss of STAG2 may be beneficial in a subset of MIBC patients. Citation Format: Nithya Krishnan, Lanni Aquila, Sofia Lage Vickers, Swathi Ramakrishnan, Kristopher Atwood, Qiang Hu, Eduardo Gomez Cortes, Jianmin Wang, Anna Woloszynska. STAG2, a novel genetic regulator of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A22.

"B" aware: Memory lane access is restricted!

Understanding the molecular mechanisms that govern the differentiation of high-affinity germinal center (GC) B cells into memory B cells versus plasma cells is a major quest of adaptive immunity. In this issue, Toboso-Navasa et al. (https://doi.org/10.1084/jem.20191933) provide evidence that the MYC-MIZ1 transcriptional repressor complex restricts the differentiation of GC B cells into MBCs.

PFKP is transcriptionally repressed by BRCA1/ZBRK1 and predicts prognosis in breast cancer.

ObjectivesThe present study aims to elucidate the underlying mechanism how PFKP is regulated by BRCA1 and the clinical significance of PFKP in breast cancer.MethodsMEF-BRCA1△/△ and the wild type counterpart MEF-BRCA1+/+ cell lines were used to test the sensitivity of glucose depletion in culture medium. Glucose Assay Kit was used to quantify glucose levels in cultural supernatant and cell lysate. Real time PCR was used to measure the mRNA expression levels of genes. Western blot was used to detect protein levels. Chromatin immunoprecipitation was used to verify the bindings between transcription factors and DNA elements. Luciferase reporter assay was performed to determine the transcriptional activity. Histochemistry assay was performed on tissue microarray.ResultsWe found that MEF-BRCA1△/△ cells consumed more glucose and were more vulnerable to glucose-deprived culture medium. The mRNA profiles and qPCR assay of MEF-BRCA1△/△ and MEF-BRCA1+/+ cells revealed that PFKP, the rate-limiting enzyme of glycolysis, was significantly upregulated in MEF-BRCA1△/△ cells. Consistently, the repressive effects of BRCA1 on PFKP were confirmed by overexpression or knockdown of BRCA1. Moreover, we also demonstrated that PFKP was suppressed by ZBRK1 as well, which was the co-repression partner of BRCA1. Mechanistically, we figured out that BRCA1 formed a transcriptional repression complex with ZBRK1 on the promoter of PFKP and consequently restrained its expression. Importantly, the expression levels of PFKP were demonstrated to associate with poor survival of patients with breast cancer.ConclusionOur study provided a new insight into the dysregulation of glycolysis in breast cancer, which might be partially due to the deficiency of BRCA1/ZBRK1 axis and subsequently reversed the transcriptional repressive effect on PFKP. We also found that PFKP overexpressed in a subset of breast cancer patients and could serve as a prognostic factor, which represented a potential target for BC therapy.

Restriction of memory B cell differentiation at the germinal center B cell positive selection stage.

Memory B cells (MBCs) are key for protection from reinfection. However, it is mechanistically unclear how germinal center (GC) B cells differentiate into MBCs. MYC is transiently induced in cells fated for GC expansion and plasma cell (PC) formation, so-called positively selected GC B cells. We found that these cells coexpressed MYC and MIZ1 (MYC-interacting zinc-finger protein 1 [ZBTB17]). MYC and MIZ1 are transcriptional activators; however, they form a transcriptional repressor complex that represses MIZ1 target genes. Mice lacking MYC-MIZ1 complexes displayed impaired cell cycle entry of positively selected GC B cells and reduced GC B cell expansion and PC formation. Notably, absence of MYC-MIZ1 complexes in positively selected GC B cells led to a gene expression profile alike that of MBCs and increased MBC differentiation. Thus, at the GC positive selection stage, MYC-MIZ1 complexes are required for effective GC expansion and PC formation and to restrict MBC differentiation. We propose that MYC and MIZ1 form a module that regulates GC B cell fate.

FRS7 and FRS12 recruit NINJA to regulate expression of glucosinolate biosynthesis genes.

The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation.

MYC-Associated Factor MAX is a Regulator of the Circadian Clock.

The circadian transcriptional network is based on a competition between transcriptional activator and repressor complexes regulating the rhythmic expression of clock-controlled genes. We show here that the MYC-associated factor X, MAX, plays a repressive role in this network and operates through a MYC-independent binding to E-box-containing regulatory regions within the promoters of circadian BMAL1 targets. We further show that this “clock” function of MAX is required for maintaining a proper circadian rhythm and that MAX and BMAL1 contribute to two temporally alternating transcriptional complexes on clock-regulated promoters. We also identified MAX network transcriptional repressor, MNT, as a fundamental partner of MAX-mediated circadian regulation. Collectively, our data indicate that MAX regulates clock gene expression and contributes to keeping the balance between positive and negative elements of the molecular clock machinery.

Pseudo Response Regulators Regulate Photoperiodic Hypocotyl Growth by Repressing PIF4/5 Transcription.

The circadian clock measures and conveys daylength information to control rhythmic hypocotyl growth in photoperiodic conditions to achieve optimal fitness, but it operates through largely unknown mechanisms. Here, we show that Pseudo Response Regulators (PRRs) coordinate with the Evening Complex (EC), a transcriptional repressor complex within the clock core oscillator, to specifically regulate photoperiodic hypocotyl growth in Arabidopsis (Arabidopsis thaliana). Intriguingly, a distinct daylength could shift the expression phase and extend the expression duration of PRRs. Multiple lines of evidence have further demonstrated that PRRs directly bind the promoters of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5 to repress their expression, hence PRRs act as transcriptional repressors of the positive growth regulators PIF4 and PIF5 Importantly, mutation or truncation of the TIMING OF CAB EXPRESSION1 (TOC1) DNA binding domain, without compromising its physical interaction with PIFs, still caused long hypocotyl growth under short days, highlighting the essential role of the PRR-PIF transcriptional module in photoperiodic hypocotyl growth. Finally, genetic analyses have demonstrated that PIF4 and PIF5 are epistatic to PRRs in the regulation of photoperiodic hypocotyl growth. Collectively, we propose that, upon perceiving daylength information, PRRs cooperate with EC to directly repress PIF4 and PIF5 transcription together with their posttranslational regulation of PIF activities, thus forming a complex regulatory network to mediate circadian clock-regulated photoperiodic growth.

Molecular mechanisms of Evening Complex activity in Arabidopsis

The Evening Complex (EC), composed of the DNA binding protein LUX ARRHYTHMO (LUX) and two additional proteins EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment, acting as an important environmental sensor and conveying this information to growth and developmental pathways. However, the molecular basis for EC DNA binding specificity and temperature-dependent activity were not known. Here, we solved the structure of the DNA binding domain of LUX in complex with DNA. Residues critical for high-affinity binding and direct base readout were determined and tested via site-directed mutagenesis in vitro and in vivo. Using extensive in vitro DNA binding assays of LUX alone and in complex with ELF3 and ELF4, we demonstrate that, while LUX alone binds DNA with high affinity, the LUX-ELF3 complex is a relatively poor binder of DNA. ELF4 restores binding to the complex. In vitro, the full EC is able to act as a direct thermosensor, with stronger DNA binding at 4 °C and weaker binding at 27 °C. In addition, an excess of ELF4 is able to restore EC binding even at 27 °C. Taken together, these data suggest that ELF4 is a key modulator of thermosensitive EC activity.

LncRNA GUARDIN suppresses cellular senescence through a LRP130-PGC1α-FOXO4-p21-dependent signaling axis.

The long noncoding RNA GUARDIN functions to protect genome stability. Inhibiting GUARDIN expression can alter cell fate decisions toward senescence or apoptosis, but the underlying molecular signals are unknown. Here, we show that GUARDIN is an essential component of a transcriptional repressor complex involving LRP130 and PGC1α. GUARDIN acts as a scaffold to stabilize LRP130/PGC1α heterodimers and their occupancy at the FOXO4 promotor. Destabilizing this complex by silencing of GUARDIN, LRP130, or PGC1α leads to increased expression of FOXO4 and upregulation of its target gene p21, thereby driving cells into senescence. We also found that GUARDIN expression was induced by rapamycin, an agent that suppresses cell senescence. FOS-like antigen 2 (FOSL2) acts as a transcriptional repressor of GUARDIN, and lower FOSL2 levels in response to rapamycin correlate with increased levels of GUARDIN. Together, these results demonstrate that GUARDIN inhibits p21-dependent senescence through a LRP130-PGC1α-FOXO4 signaling axis, and moreover, GUARDIN contributes to the anti-aging activities of rapamycin.

A40 INTESTINAL EPITHELIAL NCOR1 TARGETS TRYPTOPHAN METABOLISM AND PROTECTS AGAINST EXPERIMENTAL COLITIS

Abstract Background The nuclear co-repressor NCOR1 is a central protein that orchestrates the assembly of a large transcriptional repression complex. NCOR1 controls activation of macrophages by repressing a large variety of pro-inflammatory genes. Aims We aimed to investigate the role of intestinal epithelial NCOR1 during experimental colitis. Methods Conditional deletion of Ncor1 in the whole intestinal epithelium was achieved by crossing Villin-Cre and Ncor1loxP/loxP C57BL/6 mouse models. A gene profiling analysis in the colon of non-diseased NCOR1ΔIEC and control mice was performed. NCOR1ΔIEC and control littermate mice were treated with dextran sulfate sodium (DSS) in drinking water. Results DSS-induced colitis in NCOR1ΔIEC mice was more severe than control mice according to survival as well as clinical observations. A statistical analysis predicted 85 unique and mapped transcripts being significantly modulated between NCOR1ΔIEC and control mice. An Ingenuity Pathway Analysis from these predicted target genes identified gastrointestinal disease (79 transcripts) as top disease and biofunction. Analysis of enriched targets in specific canonical pathways predicted an increase in the tryptophan degradation pathway (P = 3.2E-02), a pathway recently demonstrated to be strongly relevant to inflammatory bowel disease severity. Indoleamine-pyrrole 2,3-dioxygenase (IDO1), that catalyzes the first and rate-limiting step of tryptophan oxidation, was induced more than 7 times in the colon of NCOR1ΔIEC mice. Induction of Ido1 was also confirmed in cultured ex vivo colon organoids deleted for Ncor1. Conclusions Our results highlight the critical role of NCOR1 to maintain intestinal inflammatory homeostasis during experimental colitis and uncover a novel function for NCOR1 in the regulation of Ido1 expression and potentially tryptophan metabolism. Funding Agencies CIHR

NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes

BackgroundAdenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear.MethodsThe dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding.ResultsPMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells.ConclusionsOur data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.

Interactome of Aiolos/Ikaros in Diffuse Large B-Cell Lymphoma (DLBCL) Reveals Novel Combination of Cereblon Modulators (CELMoD) and Histone Deacetylase (HDAC) Inhibitors

DLBCL is the most common subtype of non-Hodgkin lymphoma, constituting 30-40% of all new cases. Lenalidomide (Len) and Avadomide (Ava), small molecule cereblon modulators (CELMoD) with clinical activity in DLBCL, bind to cereblon in the CRL4CRBN E3 ligase, leading to ubiquitination and subsequent degradation of transcription factors Aiolos and Ikaros. Aiolos/Ikaros bind to the promoters of interferon stimulated genes (ISGs) and repress transcription in DLBCL cells. Degradation of these substrates by Len and Ava results in an upregulation of ISG expression leading to decreased proliferation and increased apoptosis of DLBCL cells. Ava is directly cytotoxic against ABC and GCB-DLBCL cells while Len has preferential activity in ABC-DLBCL. To date, our understanding of the COO independent activity of Ava is due to increased kinetics and greater depth of degradation of Aiolos/Ikaros compared to Len. We sought to further understand the biology of these substrates and their transcriptionally repressive activities in DLBCL. Utilizing a comprehensive approach of RNAseq and ChIP-seq of histone modifications (active: H3K27ac, H3K4me3; repressive: H3K27me3 and H3K9me3), we profiled a panel of DLBCL cell lines (2 ABC which are sensitive in proliferation assays to both Len/Ava (TMD8 and U2932), 1 GCB cell line (WSU-DLCL2) which is sensitive only to Ava and an intrinsically resistant GCB cell line (SUDHL-4)). In TMD8 and U2932 (ABC-DLBCL), both Len and Ava treatment resulted in upregulation of ISGs, while only Ava was increased ISG mRNA levels in WSU-DLCL2 (GCB-DLBCL). Neither drug induced ISG expression in the resistant SUDHL-4 (GCB-DLBCL). We found a qualitative association between activity of the drugs and baseline histone modifications of promoters for ISGs with TMD8 and U2932 being relatively enriched for H3K27ac, H3K4me3 and depleted for H3K27me3 and H3K9me3 compared to WSU-DLCL2 and SUDHL-4. SUDHL-4 had the least enrichment for poised and the most enrichment for repressive marks at the transcriptional start sites of ISGs of the cell lines profiled. These data indicate that chromatin modifications, such as decreased active histone marks and increased repressive histone marks at the promoters of ISGs correlate with Len and Ava sensitivity. We hypothesized that Aiolos/Ikaros, known substrates of Len/Ava and repressors of ISGs, were involved in regulation of nearby chromatin landscape through cooperation with transcriptional repressor complexes. To identify complexes associated with these proteins, we performed TMT labeled proteomics on nuclear lysates immunoprecipitated for Aiolos or Ikaros. We identified a significant enrichment in a number of complexes including the nucleosome remodeling deacetylase (NuRD) complex of which HDAC1 and HDAC2 are integral components. Utilizing a protein ligation assay, the interaction of Aiolos/Ikaros with multiple components of the NuRD complex including CHD4, HDAC1 and HDAC2 was confirmed. Across a panel of 10 cell lines 50-80% of cells were positive for a complex formation of Aiolos/Ikaros with a component of the NuRD complex and a majority of these cells were in the G1 phase of the cell cycle. Given the interaction of Aiolos/Ikaros with the NuRD complex, we hypothesized that a combination treatment of Ava with citarinostat (CC-241), a HDAC 1/2/3/6 inhibitor, would result in enhanced ISG expression and synergistic cell autonomous activity in DLBCL. The combination of Ava and CC-241 led to a significant increase in mRNA expression of ISGs such as IFIT3 by 4.2-14 fold and DDX58 by 6.8-8.2 fold compared to either single agent (IFIT3: Ava 0.5-8 fold; CC-241 1.5-1.6 fold p<0.05-0.001; DDX58: Ava 1.5-3 fold; CC-241 2.2-3 fold p<0.05-0.01). CC-241 demonstrated an inhibition of cell proliferation and increased apoptosis in a dose dependent and COO independent manner (1-4 mM) in a panel of DLBCL cell lines. The combination resulted in a synergistic anti-proliferative activity against 6 of 11 DLBCL cell lines profiled. These data demonstrate that Aiolos/Ikaros regulate ISG expression in DLBCL in part through directing epigenetic modifications of their promoters. The combination of Ava with CC-241 results in a synergistic inhibition of DLBCL cell proliferation, through enhanced derepression of transcriptional activity at the promoter regions of ISGs. These data highlight the potential of combining avadomide with a HDAC inhibitor such as citarinostat in DLBCL. Disclosures Hagner: Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Chiu:Celgene: Employment, Equity Ownership, Patents & Royalties. Chopra:Amgen Inc: Employment; Celgene Corporation: Equity Ownership. Colombo:Celgene: Employment, Equity Ownership. Patel:Celgene: Equity Ownership. Ortiz:Celgene Corporation: Employment, Equity Ownership. Loos:Celgene: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Waldman:Celgene: Employment, Equity Ownership, Patents & Royalties. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

Arabidopsis ECAP Is a New Adaptor Protein that Connects JAZ Repressors with the TPR2 Co-repressor to Suppress Jasmonate-Responsive Anthocyanin Accumulation

Suppression mechanisms mediated by transcriptional repressors commonly exist in diverse phytohormone signaling pathways. In Arabidopsis thaliana, JASMONATE-ZIM DOMAIN (JAZ) proteins are transcriptional repressors that function as negative regulators of diverse JA responses. Novel Interactor of JAZ (NINJA) is an adaptor protein connecting JAZs with the co-repressor, TOPLESS (TPL), to mediate gene repression in JA-dependent root growth inhibition and defense pathways. However, whether NINJA or other adaptor proteins are employed in other JA-responsive biological processes remains to be elucidated. In the present study, we demonstrate that a previously uncharacterized protein, ECAP (EAR motif-Containing Adaptor Protein), directly interacts with JAZ6 and JAZ8 and enhances their transcriptional repression activities. We provide evidence that ECAP is a novel adaptor protein for JAZ6/8 recruitment of the transcriptional co-repressor, TOPLESS-RELATED 2 (TPR2), into a transcriptional repressor complex that represses the WD-repeat/bHLH/MYB complex, an important transcriptional activator in the JA-dependent anthocyanin biosynthesis pathway. Our findings, together with previous reports, reveal that specific adaptor proteins play a critical role in distinct JA responses by pairing different JAZs (which possess overlapping but also specific functions) with the general co-repressors, TPL and TPRs.

ZBTB7A Mediates the Transcriptional Repression Activity of the Androgen Receptor in Prostate Cancer.

Loss of expression of context-specific tumor suppressors is a critical event that facilitates the development of prostate cancer. Zinc finger and BTB domain containing transcriptional repressors, such as ZBTB7A and ZBTB16, have been recently identified as tumor suppressors that play important roles in preventing prostate cancer progression. In this study, we used combined ChIP-seq and RNA-seq analyses of prostate cancer cells to identify direct ZBTB7A-repressed genes, which are enriched for transcriptional targets of E2F, and identified that the androgen receptor (AR) played a critical role in the transcriptional suppression of these E2F targets. AR recruitment of the retinoblastoma protein (Rb) was required to strengthen the E2F-Rb transcriptional repression complex. In addition, ZBTB7A was rapidly recruited to the E2F-Rb binding sites by AR and negatively regulated the transcriptional activity of E2F1 on DNA replication genes. Finally, ZBTB7A suppressed the growth of castration-resistant prostate cancer (CRPC) in vitro and in vivo, and overexpression of ZBTB7A acted in synergy with high-dose testosterone treatment to effectively prevent the recurrence of CRPC. Overall, this study provides novel molecular insights of the role of ZBTB7A in CRPC cells and demonstrates globally its critical role in mediating the transcriptional repression activity of AR. SIGNIFICANCE: ZBTB7A is recruited to the E2F-Rb binding sites by AR and negatively regulates the transcriptional activity of E2F1 on DNA replication genes.

Changes in chromatin accessibility ensure robust cell cycle exit in terminally differentiated cells.

During terminal differentiation, most cells exit the cell cycle and enter into a prolonged or permanent G0 in which they are refractory to mitogenic signals. Entry into G0 is usually initiated through the repression of cell cycle gene expression by formation of a transcriptional repressor complex called dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM). However, when DREAM repressive function is compromised during terminal differentiation, additional unknown mechanisms act to stably repress cycling and ensure robust cell cycle exit. Here, we provide evidence that developmentally programmed, temporal changes in chromatin accessibility at a small subset of critical cell cycle genes act to enforce cell cycle exit during terminal differentiation in the Drosophila melanogaster wing. We show that during terminal differentiation, chromatin closes at a set of pupal wing enhancers for the key rate-limiting cell cycle regulators Cyclin E (cycE), E2F transcription factor 1 (e2f1), and string (stg). This closing coincides with wing cells entering a robust postmitotic state that is strongly refractory to cell cycle reactivation, and the regions that close contain known binding sites for effectors of mitogenic signaling pathways such as Yorkie and Notch. When cell cycle exit is genetically disrupted, chromatin accessibility at cell cycle genes remains unaffected, and the closing of distal enhancers at cycE, e2f1, and stg proceeds independent of the cell cycling status. Instead, disruption of cell cycle exit leads to changes in accessibility and expression of a subset of hormone-induced transcription factors involved in the progression of terminal differentiation. Our results uncover a mechanism that acts as a cell cycle–independent timer to limit the response to mitogenic signaling and aberrant cycling in terminally differentiating tissues. In addition, we provide a new molecular description of the cross talk between cell cycle exit and terminal differentiation during metamorphosis.

Dopamine D1 receptor (D1R) expression is controlled by a transcriptional repressor complex containing DISC1

Disrupted-in-Schizophrenia 1 (DISC1) is a scaffold protein implicated in various psychiatric diseases. Dysregulation of the dopamine system has been associated with DISC1 deficiency, while the molecular mechanism is unclear. In this study, we propose a novel molecular mechanism underlying the transcriptional regulation of the dopamine D1 receptor (D1R) in the striatum via DISC1. We verified the increase in D1R at the transcriptional level in the striatum of DISC1-deficient mouse models and altered histone acetylation status at the D1r locus. We identified a functional interaction between DISC1 and Kruppel-like factor 16 (KLF16). KLF16 translocates DISC1 into the nucleus and forms a regulatory complex by recruiting SIN3A corepressor complexes to the D1r locus. Moreover, DISC1-deficient mice have altered D1R-mediated signaling in the striatum and exhibit hyperlocomotion in response to cocaine; the blockade of D1R suppresses these effects. Taken together, our results suggest that nuclear DISC1 plays a critical role in the transcriptional regulation of D1R in the striatal neuron, providing a mechanistic link between DISC1 and dopamine-related psychiatric symptoms.

The ATPase BRG1/SMARCA4is a protein interaction platform that recruits BAF subunits and the transcriptional repressor REST/NRSF in neural progenitor cells.

The BAF complex (SWI/SNF) is an ATP-dependent chromatin remodeler that adapts the structural organization of the chromatin. Despite a growing understanding of the composition of BAF in different cell types, the interaction network within the BAF complex is poorly understood. Here, we characterized an isoform of the BRG1/SMARCA4 ATPase expressed in human neural progenitor cells. By electron microscopy and image processing, the neural BRG1/SMARCA4 shows an elongated globular structure, which provides a considerably larger surface than anticipated. We show that neural BRG1/SMARCA4 binds to BAF57/SMARCE1 and BAF60A/SMARCD1, two further components of BAF. Moreover, we demonstrate an interaction between the neural BRG1/SMARCA4 isoform and the central neurodevelopmental transcriptional repressor REST/NRSF. Our results provide insights into the assembly of a central transcriptional repressor complex, link the structure of the neural BRG1/SMARCA4 to its role as a protein-protein interaction platform and suggest BRG1/SMARCA4 as a key determinant that directs the BAF complex to specific DNA sites by interacting with transcription factors and regulators.

DREAM and RB cooperate to induce gene repression and cell-cycle arrest in response to p53 activation.

Most human cancers acquire mutations causing defects in the p53 signaling pathway. The tumor suppressor p53 becomes activated in response to genotoxic stress and is essential for arresting the cell cycle to facilitate DNA repair or to initiate apoptosis. p53-induced cell cycle-arrest is mediated by expression of the CDK inhibitor p21WAF1/Cip1, which prevents phosphorylation and inactivation of the pocket proteins RB, p130, and p107. In a hypophosphorylated state, pocket proteins bind to E2F factors forming RB-E2F and DREAM transcriptional repressor complexes. Here, we analyze the influence of RB and DREAM on p53-induced gene repression and cell-cycle arrest. We show that abrogation of DREAM function by knockout of the DREAM component LIN37 results in a reduced repression of cell-cycle genes. We identify the genes repressed by the p53-DREAM pathway and describe a set of genes that is downregulated by p53 independent of LIN37/DREAM. Most strikingly, p53-dependent repression of cell-cycle genes is completely abrogated in LIN37−/−;RB−/− cells leading to a loss of the G1/S checkpoint. Taken together, we show that DREAM and RB are key factors in the p53 signaling pathway to downregulate a large number of cell-cycle genes and to arrest the cell cycle at the G1/S transition.

RNA-INTERFERENCE SCREEN IDENTIFIES MTA1 AS A REGULATOR OF HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

The hematopoietic stem cell (HSC) is arguably one of the most well studied adult stem cells, however, the regulatory mechanisms that govern HSC fate are still not fully understood. In order to identify novel genetic regulators of HSC function, we used a near-genome wide RNA-interference (RNAi) lentivirus library to target primary human hematopoietic stem and progenitor cells (HSPCs). Among other candidates, we identified metastasis-associated 1 (MTA1) as a negative regulator of human HSPC propagation in vitro. MTA1 is part of the nucleosome remodeling deacetylase (NuRD) complex, a pan-cellular transcriptional repression complex, similar to other chromatin remodelers like SWI/SNF and the Polycomb complexes. Upon knockdown of MTA1 by independent shRNAs in primary human cord blood CD34+ cells, we observed an increased cell expansion during culture and a relative accumulation of immature CD34+CD90+ cells compared to control. By contrast, the colony forming potential was reduced, indicating perturbed differentiation and ability to form mature blood cells. Furthermore, transplantation experiments in immunodeficient mice showed a significant reduction of human chimerism in both blood and bone marrow from HSPCs with knockdown of MTA1. These findings suggest that MTA1 regulates the balance between renewal and differentiation of human HSPCs, as well as their in vivo engraftment potential. To gain more molecular insights we performed global transcriptional profiling and found a strong downregulation of essential genes related to cell engraftment and adhesion, including CXCR4, which might, at least in part, explain the engraftment defect seen upon MTA1 knockdown. Current studies are aimed at exploring the interaction between MTA1 and other NuRD complex members, such as CHD4, and how their interplay regulates transcriptional programs involved in human HSC biology.

Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ

NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.