Integrated complex coregulatory networks together modulate, reorganize, and fine-tune patterns of transcriptional response to integrate multiple signaling pathways. These studies have begun to define the sequence signatures for combinatorial codes that regulate specific cohorts of gene targets. Rapid nuclear motor–dependent regulation of nuclear architecture and noncoding RNAs that act as "sensors" are uncovering some of the genome-wide mechanisms used to mediate complex transcriptional response programs required for all metazoans.
Function of a Repressor "Checkpoint" in Regulated Programs of Gene Expression
We have found an unexpected strategy that is based, in part, on a requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) that modify histone H3K9, as well as methylation of other regulatory targets, imposing strict gene-specific "gatekeeper" functions. This prevents unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters, causing constitutive gene activation in the absence of stimulating signals. We used a conditional gene deletion strategy to investigate functions of LSD1 in development. LSD1, a component of the CoREST/CtBP-containing corepressor complex, is required for late cell-lineage determination and differentiation during pituitary organogenesis, acting primarily on target gene activation programs.
Repression Checkpoints in Progression from Neural Stem Cell to Neuron
Our investigation of corepressors that are important in maintenance of the neural stem cell state identified distinct critical roles for the related corepressors NCoR, required for active repression of the glial pathway, and SMRT, important in forebrain development and in maintenance of the neural stem cell state. SMRT proved to be required for the actions of both retinoic acid–dependent and Notch-dependent forebrain development, and is required both for prevention of retinoic acid receptor–dependent induction of differentiation along a neuronal pathway in the absence of ligand. Our data revealed that SMRT represses expression of the jumonji-domain containing gene JMJD3, acting as a histone H3 trimethyl K27 demethylase capable of activating specific components of the neurogenic program. We identified a previously unrecognized functional interaction between SMRT and the forkhead protein FOXP1. This SMRT:FOXP interaction proved to be required for physiological cardiac growth and regulation of macrophage differentiation. This suggests that SMRT and FOXP1 define a functional biological unit required to orchestrate specific programs critical for mammalian organogenesis.
Inflammation and Disease
Understanding the regulatory interactions between inflammation and transcriptional pathways provides new approaches to diverse disease. We have found that prostate cancer tissue exhibits specific macrophage–cancer cell interactions and that these interactions mediate a switch in SARM (selective androgen receptor modulator) function from repression to activation in vivo. This switch is based on the selective presence of an evolutionarily conserved receptor amino-terminal helical motif in a sex steroid receptor that modulates recruitment of a component of the NCoR corepressor complex TAB2, which serves as a molecular beacon for recruitment of a protein kinase (MEKK1) that mediates dismissal of the NCoR complex. Peptides corresponding to the helical motif of androgen receptor or estrogen receptor-α can block macrophage-dependent resistance, which has led to potential new therapeutic approaches for treatment of prostate cancer.
Nuclear Receptor Exchange Functions in Gene Regulation
Our studies have revealed that many signaling pathways, including Notch, NFκB, and nuclear receptor ligands, are subjected to a dual-repression checkpoint. This combinatoric strategy serves to prevent illegitimate signal-independent activation to achieve regulated gene expression by imposing robust control on the dismissal of corepressors. This requires the release of both CtBP1/2- and NCoR/SMRT-dependent repression, through the coordinate action of two highly related exchange factors, the transducer β-like proteins TBL1 and TBLR1, which license ubiquitylation and degradation of CtBP1/2 and NCoR/SMRT, respectively. Intriguingly, TBL1 and TBLR1 function and differential specificity reside in five Ser/Thr phosphorylation site differences, each regulated directly by phosphorylation at the level of the target gene promoter. We found a SUMOylation-dependent mechanism for transrepression was required for both the PPARγ and LXR nuclear receptors to negatively regulate overlapping, but distinct, subsets of proinflammatory genes, differentially regulating complex programs of gene expression that control immunity and homeostasis.
Histone H2A Monoubiquitination/Deubiquitination Programs
We have observed that the NCoR/histone deacetylase complex recruits a specific histone H2A ubiquitin ligase, referred to as 2A-HUB, to a subset of regulated gene promoters, catalyzing monoubiquitination of H2A at lysine 119 and mediating selective repression of a specific set of chemokine genes in macrophages. We identified a novel H2A deubiquitinase (2A-DUB)specific for monoubiquitinated H2A (uH2A), which regulates transcription by coordinating histone acetylation and deubiquitination and destabilizing the association of linker histone H1 with nucleosomes. 2A-DUB interacts with the p/CAF histone acetyltransferase in a coregulatory protein complex; its deubiquitinase activity is modulated by the status of acetylation of nucleosomal histones and participates in transcriptional regulation events in androgen receptor–dependent gene activation, serving as a prostate cancer–related mark. We suggest that H2A ubiquitination is a widely used mechanism for many regulatory transcriptional programs that use distinct receptor H2A ubiquitin ligases/deubiquitinases.
Architectural Strategies in Regulated Transcriptional Programs
Using a (ChIP-DSL) DNA selection and ligation (DSL) strategy, developed with Xiang-Dong Fu (University of California, San Diego), we have profiled general and sequence-specific DNA-binding transcription factors, initially focusing on estrogen and androgen receptors. We identified tissue-specific transcription of a retrotransposon repeat in the murine growth hormone locus that appears to be required for gene activation. This repeat may serve as a "boundary" to block the influence of repressive chromatin modifications, generating short, overlapping Pol II– and Pol III–driven transcripts, both of which are necessary to restructure the regulated locus into nuclear compartments. Our data suggest that transcription of specific interspersed repetitive sequences may function as a developmental and homeostatic strategy for the establishment of functionally distinct domains within the mammalian genome.
We have found that estrogen or androgen receptor ligands can induce rapid interchromosomal interactions among subsets of estrogen receptor-α–bound transcription units, with a dramatic reorganization of nuclear territories requiring the actions of nuclear actin/myosin-I motor, and dynein light chain 1 (DLC1). The histone lysine demethylase LSD1 proved to be required for directing interactions between specific interchromosomal interaction loci to distinct interchromatin granules, thought to be "storage" sites for splicing machinery, which suggests that three-dimensional, motor-dependent interactions were required to achieve enhanced transcription of specific estrogen receptor target genes. These findings suggest a broader role of signal-dependent changes in nuclear architecture in the orchestration of regulated gene expression, and possibly tumor translocation events in the mammalian cells.
Noncoding RNAs and Genotoxic Stress
Our studies in collaboration with Christopher Glass (University of California, San Diego) and Riki Kurokawa (Saitama Medical University, Japan) have revealed that the RNA-binding protein TLS (translocation liposarcoma) is a key transcriptional regulatory sensor of DNA damage signals. Based on its allosteric modulation by RNA, TLS specifically binds to and inhibits CBP/p300 HAT activities on a repressed gene target, cyclin D1 (CCND1). This recruitment of TLS to the CCND1 promoter is directed by single-stranded, low-copy-number, noncoding RNA transcripts on the 5' regulatory regions of CCND1 that are induced in response to DNA damage signals. Our data suggest that signal-induced noncoding RNAs can act cooperatively as selective ligands, recruiting and modulating the activities of distinct classes of promoter-associated RNA-binding coregulators in response to specific signals, to integrate transcriptional programs.
