Charles Y. Lin

Impact of the gut microbiota on enhancer accessibility in gut intraepithelial lymphocytes

Significance Comparing germ-free mice with those colonized at birth or later provides a way to determine how gut microbial community exposure affects the chromatin landscape of cells along the gut or at remote sites, ascertain how alterations in chromatin accessibility are correlated with functional features of different lineages, and determine whether there is a critical window of exposure when microbial signals must be received to alter the landscape durably. Genome-wide analysis of chromatin accessibility in intraepithelial lymphocytes and circulating T cells purified from gnotobiotic mice revealed enhancers and flanking genes involved in signaling and metabolic pathways that are sensitive to colonization status. Colonization does not fundamentally alter lineage-specific cis-regulatory landscapes but induces quantitative changes in the accessibility of preestablished enhancer elements. The gut microbiota impacts many aspects of host biology including immune function. One hypothesis is that microbial communities induce epigenetic changes with accompanying alterations in chromatin accessibility, providing a mechanism that allows a community to have sustained host effects even in the face of its structural or functional variation. We used Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) to define chromatin accessibility in predicted enhancer regions of intestinal αβ+ and γδ+ intraepithelial lymphocytes purified from germ-free mice, their conventionally raised (CONV-R) counterparts, and mice reared germ free and then colonized with CONV-R gut microbiota at the end of the suckling–weaning transition. Characterizing genes adjacent to traditional enhancers and super-enhancers revealed signaling networks, metabolic pathways, and enhancer-associated transcription factors affected by the microbiota. Our results support the notion that epigenetic modifications help define microbial community-affiliated functional features of host immune cell lineages.

Transcriptional landscape of the human cell cycle

Significance Our study provided a comprehensive view of the transcriptional landscape across the cell cycle. We revealed lag between transcription and steady-state RNA expression at the cell-cycle level and characterized a large amount of active transcription during early mitosis. In addition, our analysis identified thousands of enhancer RNAs and related transcription factors that are highly correlated with cell-cycle–regulated transcription but not with steady-state expression, thus highlighting the importance of transcriptional and epigenetic dynamics during cell-cycle progression. Steady-state gene expression across the cell cycle has been studied extensively. However, transcriptional gene regulation and the dynamics of histone modification at different cell-cycle stages are largely unknown. By applying a combination of global nuclear run-on sequencing (GRO-seq), RNA sequencing (RNA-seq), and histone-modification Chip sequencing (ChIP-seq), we depicted a comprehensive transcriptional landscape at the G0/G1, G1/S, and M phases of breast cancer MCF-7 cells. Importantly, GRO-seq and RNA-seq analysis identified different cell-cycle–regulated genes, suggesting a lag between transcription and steady-state expression during the cell cycle. Interestingly, we identified genes actively transcribed at early M phase that are longer in length and have low expression and are accompanied by a global increase in active histone 3 lysine 4 methylation (H3K4me2) and histone 3 lysine 27 acetylation (H3K27ac) modifications. In addition, we identified 2,440 cell-cycle–regulated enhancer RNAs (eRNAs) that are strongly associated with differential active transcription but not with stable expression levels across the cell cycle. Motif analysis of dynamic eRNAs predicted Kruppel-like factor 4 (KLF4) as a key regulator of G1/S transition, and this identification was validated experimentally. Taken together, our combined analysis characterized the transcriptional and histone-modification profile of the human cell cycle and identified dynamic transcriptional signatures across the cell cycle.

Signal-Dependent Recruitment of BRD4 to Cardiomyocyte Super-Enhancers Is Suppressed by a MicroRNA

BRD4 governs pathological cardiac gene expressionxa0by binding acetylated chromatin, resulting in enhanced RNA polymerase II (Pol II) phosphorylation and transcription elongation. Here, we describe a signal-dependent mechanism for the regulation of BRD4 in cardiomyocytes. BRD4 expression is suppressed by microRNA-9 (miR-9), which targets the 3 UTR of the Brd4 transcript. In response to stress stimuli, miR-9 is downregulated, leading to derepression of BRD4 and enrichment of BRD4 at long-range super-enhancers (SEs) associated with pathological cardiac genes. A miR-9 mimic represses stimulus-dependent targeting of BRD4 to SEs and blunts Polxa0II phosphorylation at proximal transcription start sites, without affecting BRD4 binding to SEs that control constitutively expressed cardiac genes. These findings suggest that dynamic enrichment of BRD4 at SEs genome-wide serves a crucial role in the control of stress-induced cardiac gene expression and define a miR-dependent signaling mechanism for the regulation of chromatin state and Pol II phosphorylation.

Combinatorial inhibition of PTPN12-regulated receptors leads to a broadly effective therapeutic strategy in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer diagnosed in more than 200,000 women each year and is recalcitrant to targeted therapies. Although TNBCs harbor multiple hyperactive receptor tyrosine kinases (RTKs), RTK inhibitors have been largely ineffective in TNBC patients thus far. We developed a broadly effective therapeutic strategy for TNBC that is based on combined inhibition of receptors that share the negative regulator PTPN12. Previously, we and others identified the tyrosine phosphatase PTPN12 as a tumor suppressor that is frequently inactivated in TNBC. PTPN12 restrains several RTKs, suggesting that PTPN12 deficiency leads to aberrant activation of multiple RTKs and a co-dependency on these receptors. This in turn leads to the therapeutic hypothesis that PTPN12-deficient TNBCs may be responsive to combined RTK inhibition. However, the repertoire of RTKs that are restrained by PTPN12 in human cells has not been systematically explored. By methodically identifying the suite of RTK substrates (MET, PDGFRβ, EGFR, and others) inhibited by PTPN12, we rationalized a combination RTK-inhibitor therapy that induced potent tumor regression across heterogeneous models of TNBC. Orthogonal approaches revealed that PTPN12 was recruited to and inhibited these receptors after ligand stimulation, thereby serving as a feedback mechanism to limit receptor signaling. Cancer-associated mutation of PTPN12 or reduced PTPN12 protein levels diminished this feedback mechanism, leading to aberrant activity of these receptors. Restoring PTPN12 protein levels restrained signaling from RTKs, including PDGFRβ and MET, and impaired TNBC survival. In contrast with single agents, combined inhibitors targeting the PDGFRβ and MET receptors induced the apoptosis in TNBC cells in vitro and in vivo. This therapeutic strategy resulted in tumor regressions in chemo-refractory patient-derived TNBC models. Notably, response correlated with PTPN12 deficiency, suggesting that impaired receptor feedback may establish a combined addiction to these proto-oncogenic receptors. Taken together, our data provide a rationale for combining RTK inhibitors in TNBC and other malignancies that lack receptor-activating mutations.

BET bromodomain proteins regulate enhancer function during adipogenesis

Significance Adipocyte differentiation is necessary for metabolic homeostasis. Transcription factors control adipocyte differentiation by regulating expression of genes required for adipocyte function. Here we show that the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins are important coregulators of adipogenesis. BRD4 binds to enhancers that drive expression of PPARγ, a master transcription factor required for normal adipocyte differentiation. Disrupting BRD4 at these enhancers blocks PPARγ expression and impairs differentiation. Understanding the molecular determinants of PPARγ expression and adipocyte differentiation may provide insights into the pathogenesis of metabolic diseases related to adipose tissue dysfunction and obesity. Furthermore, these data implicate metabolic pathways and their transcriptional regulators as potential targets of BET bromodomain inhibitors, as currently under investigation in human clinical trials of cancer therapy. Developmental transitions are guided by master regulatory transcription factors. During adipogenesis, a transcriptional cascade culminates in the expression of PPARγ and C/EBPα, which orchestrate activation of the adipocyte gene expression program. However, the coactivators controlling PPARγ and C/EBPα expression are less well characterized. Here, we show the bromodomain-containing protein, BRD4, regulates transcription of PPARγ and C/EBPα. Analysis of BRD4 chromatin occupancy reveals that induction of adipogenesis in 3T3L1 fibroblasts provokes dynamic redistribution of BRD4 to de novo super-enhancers proximal to genes controlling adipocyte differentiation. Inhibition of the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins impedes BRD4 occupancy at these de novo enhancers and disrupts transcription of Pparg and Cebpa, thereby blocking adipogenesis. Furthermore, silencing of these BRD4-occupied distal regulatory elements at the Pparg locus by CRISPRi demonstrates a critical role for these enhancers in the control of Pparg gene expression and adipogenesis in 3T3L1s. Together, these data establish BET bromodomain proteins as time- and context-dependent coactivators of the adipocyte cell state transition.

Mutant NPM1 Maintains the Leukemic State through HOX Expression

NPM1 is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (AML). In AML cells, NPM1 mutations result in abnormal cytoplasmic localization of the mutant protein (NPM1c); however, it is unknown whether NPM1c is required to maintain the leukemic state. Here, we show that loss of NPM1c from the cytoplasm, either through nuclear relocalization or targeted degradation, results in immediate downregulation of homeobox (HOX) genes followed by differentiation. Finally, we show that XPO1 inhibition relocalizes NPM1c to the nucleus, promotes differentiation of AML cells, and prolongs survival of Npm1-mutated leukemic mice. We describe an exquisite dependency of NPM1-mutant AML cells on NPM1c, providing the rationale for the use of nuclear export inhibitors in AML with mutated NPM1.

NRL and CRX Define Photoreceptor Identity and Reveal Subgroup-Specific Dependencies in Medulloblastoma

Cancer cells often express differentiation programs unrelated to their tissue of origin, although the contribution of these aberrant phenotypes to malignancy is poorly understood. An aggressive subgroup of medulloblastoma, a malignant pediatric brain tumor of the cerebellum, expresses a photoreceptor differentiation program normally expressed in the retina. We establish that two photoreceptor-specific transcription factors, NRL and CRX, are master regulators of this program and are required for tumor maintenance in this subgroup. Beyond photoreceptor lineage genes, we identify BCL-XL as a key transcriptional target of NRL and provide evidence substantiating anti-BCL therapy as a rational treatment opportunity for select MB patients. Our results highlight the utility of studying aberrant differentiation programs in cancer and their potential as selective therapeutic vulnerabilities.

Abstract 5490: Non-overlapping promoter and super-enhancer-associated dependencies in multiple myeloma

The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well-defined. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators to identify their individual contribution to eventual functional effect in multiple myeloma (MM). To better understand how E2F1 and DP1 drive proliferation, we mapped the global occupancy of E2F1/DP1 in MM. Integration of E2F1 and DP1 genomic localization to MM reference epigenome revealed specific co-occupancy of the factors at promoters of active genes marked by H3K4me3, with a strong positive correlation between E2F and RNA Polymerase II (RNA Pol II) binding at transcription start sites. In contrast, active enhancers, as defined by promoter distal Mediator (MED1) peaks and marked by H3K27ac and BRD4, showed virtually no E2F binding. Unbiased hierarchical clustering revealed distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth/proliferation genes and BETs disproportionately at enhancer- regulated tissue specific genes confirming that these factors establish distinct target gene programs. At the extremes, we found less than 10% of genes were among the top 500 in BRD4 enhancer signal (i.e. SE-regulated) and top 500 E2F promoter signal. We hypothesized that the presence of BETs and E2F in distinct regulatory axes divides active genes in MM into those that can be selectively influenced by BET inhibition or E2F perturbation, but not both. In line with this we have observed that dual E2F and BET inhibition is synergistic for MM cell growth, both in vitro and in vivo. In conclusions, our results highlights the existence of distinct regulatory axes controlled by promoter and enhancer driven processes, suggesting a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy. Citation Format: Mariateresa Fulciniti, Charles Y. Lin, Mehmet K. Samur, Richard Young, Herve Avet-loiseau, Kenneth Anderson, James Bradner, Nikhil Munshi. Non-overlapping promoter and super-enhancer-associated dependencies in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5490. doi:10.1158/1538-7445.AM2017-5490