Neuronal MAPT expression is mediated by long-range interactions with cis-regulatory elements.

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC & Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

Computational resources to define alleles and altered regulatory motifs at genomically edited candidate response elements.

Unequivocal functional assessment of candidate genomic regulatory regions, such as transcriptional response elements, requires genetic alteration at their native chromosomal loci. Targeted DNA cleavage by Cas9 or other programmable nucleases enables analysis at virtually any genomic region, and diverse alleles generated by editing can be defined by deep sequencing for functional analysis. Interpretation of disrupted response elements, however, presents a special challenge, as these regions typically comprise clustered DNA binding motifs for multiple transcriptional regulatory factors (TFs); DNA sequence differences, natural or engineered, that affect binding by one TF can confer loss or gain of binding sites for other TFs. To address these and other analytical complexities, we created three computational tools that together integrate, in a single experiment, allele definition and TF binding motif evaluation for up to 9216 clones isolated, sequenced and propagated from Cas9-treated cell populations. We demonstrate 1) the capacity to functionally assess edited TF binding sites to query response element function, and 2) the efficacy and utility of these tools, by analyzing cell populations targeted by Cas9 for disruption of example glucocorticoid receptor (GR) binding motifs near FKBP5, a GR-regulated gene in the human adenocarcinoma cell line A549.

Glucocorticoid receptor control of transcription: precision and plasticity via allostery.

The glucocorticoid receptor (GR) is a constitutively expressed transcriptional regulatory factor (TRF) that controls many distinct gene networks, each uniquely determined by particular cellular and physiological contexts. The precision of GR-mediated responses seems to depend on combinatorial, context-specific assembly of GR-nucleated transcription regulatory complexes at genomic response elements. In turn, evidence suggests that context-driven plasticity is conferred by the integration of multiple signals, each serving as an allosteric effector of GR conformation, a key determinant of regulatory complex composition and activity. This structural and mechanistic perspective on GR regulatory specificity is likely to extend to other eukaryotic TRFs.

Mediator and post-recruitment regulation of RNA polymerase II.

The human Mediator complex interacts extensively with the RNA polymerase II (Pol II) enzyme and recent data from our lab suggest activator-induced structural shifts within Mediator trigger activation of stalled Pol II. These results are discussed together with other recent findings regarding post-recruitment regulation of Pol II.

The human CDK8 subcomplex is a molecular switch that controls Mediator coactivator function.

The human CDK8 subcomplex (CDK8, cyclin C, Med12, and Med13) negatively regulates transcription in ways not completely defined; past studies suggested CDK8 kinase activity was required for its repressive function. Using a reconstituted transcription system together with recombinant or endogenous CDK8 subcomplexes, we demonstrate that, in fact, Med12 and Med13 are critical for subcomplex-dependent repression, whereas CDK8 kinase activity is not. A hallmark of activated transcription is efficient reinitiation from promoter-bound scaffold complexes that recruit a series of pol II enzymes to the gene. Notably, the CDK8 submodule strongly represses even reinitiation events, suggesting a means to fine tune transcript levels. Structural and biochemical studies confirm the CDK8 submodule binds the Mediator leg/tail domain via the Med13 subunit, and this submodule-Mediator association precludes pol II recruitment. Collectively, these results reveal the CDK8 subcomplex functions as a simple switch that controls the Mediator-pol II interaction to help regulate transcription initiation and reinitiation events. As Mediator is generally required for expression of protein-coding genes, this may reflect a common mechanism by which activated transcription is shut down in human cells.

The human CDK8 subcomplex is a histone kinase that requires Med12 for activity and can function independently of mediator.

The four proteins CDK8, cyclin C, Med12, and Med13 can associate with Mediator and are presumed to form a stable "CDK8 subcomplex" in cells. We describe here the isolation and enzymatic activity of the 600-kDa CDK8 subcomplex purified directly from human cells and also via recombinant expression in insect cells. Biochemical analysis of the recombinant CDK8 subcomplex identifies predicted (TFIIH and RNA polymerase II C-terminal domain [Pol II CTD]) and novel (histone H3, Med13, and CDK8 itself) substrates for the CDK8 kinase. Notably, these novel substrates appear to be metazoan-specific. Such diverse targets imply strict regulation of CDK8 kinase activity. Along these lines, we observe that Mediator itself enables CDK8 kinase activity on chromatin, and we identify Med12--but not Med13--to be essential for activating the CDK8 kinase. Moreover, mass spectrometry analysis of the endogenous CDK8 subcomplex reveals several associated factors, including GCN1L1 and the TRiC chaperonin, that may help control its biological function. In support of this, electron microscopy analysis suggests TRiC sequesters the CDK8 subcomplex and kinase assays reveal the endogenous CDK8 subcomplex--unlike the recombinant submodule--is unable to phosphorylate the Pol II CTD.

Cooperative activity of cdk8 and GCN5L within Mediator directs tandem phosphoacetylation of histone H3.

The human Mediator complex is generally required for expression of protein-coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex. Consequently, this derivative 'T/G-Mediator' complex does not directly activate transcription in a reconstituted human transcription system. However, within T/G-Mediator, cdk8 phosphorylates serine-10 on histone H3, which in turn stimulates H3K14 acetylation by GCN5L within the complex. Tandem phosphoacetylation of H3 correlates with transcriptional activation, and ChIP assays demonstrate co-occupancy of T/G-Mediator components at several activated genes in vivo. Moreover, cdk8 knockdown causes substantial reduction of global H3 phosphoacetylation, suggesting that T/G-Mediator is a major regulator of this H3 mark. Cooperative H3 modification provides a mechanistic basis for GCN5L association with cdk8-Mediator and also identifies a biochemical means by which cdk8 can indirectly activate gene expression. Indeed our results suggest that T/G-Mediator directs early events-such as modification of chromatin templates-in transcriptional activation.

Control of cell cycle-dependent degradation of c-Ski proto-oncoprotein by Cdc34.

It is known that excess amounts of Ski, or any member of its proto-oncoprotein family, causes disruption of the transforming growth factor beta signal transduction pathway, thus causing oncogenic transformation of cells. Previous studies indicate that Ski is a relatively unstable protein whose expression levels can be regulated by ubiquitin-mediated proteolysis. Here, we investigate the mechanism by which the stability of Ski is regulated. We show that the steady-state levels of Ski protein are controlled post-translationally by cell cycle-dependent proteolysis, wherein Ski is degraded during the interphase of the cell cycle but is relatively stable during mitosis. Furthermore, we demonstrate that the ubiquitin-conjugating enzyme Cdc34 mediates cell cycle-dependent Ski degradation both in vitro and in vivo. Overexpression of dominant-negative Cdc34 stabilizes Ski and enhances its ability to antagonize TGF-beta signaling. Our data suggest that regulated proteolysis of Ski is one of the key mechanisms that control the threshold levels of this proto-oncoprotein, and thus prevents epithelial cells from becoming TGF-beta resistant.