Multiple structural maintenance of chromosome complexes at transcriptional regulatory elements.

Transcription factors control cell-specific gene expression programs by binding regulatory elements and recruiting cofactors and the transcription apparatus to the initiation sites of active genes. One of these cofactors is cohesin, a structural maintenance of chromosomes (SMC) complex that is necessary for proper gene expression. We report that a second SMC complex, condensin II, is also present at transcriptional regulatory elements of active genes during interphase and is necessary for normal gene activity. Both cohesin and condensin II are associated with genes in euchromatin and not heterochromatin. The two SMC complexes and the SMC loading factor NIPBL are particularly enriched at super-enhancers, and the genes associated with these regulatory elements are especially sensitive to reduced levels of these complexes. Thus, in addition to their well-established functions in chromosome maintenance during mitosis, both cohesin and condensin II make important contributions to the functions of the key transcriptional regulatory elements during interphase.

Chromatin organization and transcriptional regulation.

Cell type specific transcriptional regulation must be adhered to in order to maintain cell identity throughout the lifetime of an organism, yet it must be flexible enough to allow for responses to endogenous and exogenous stimuli. This regulation is mediated not only by molecular factors (e.g. cell type specific transcription factors, histone and DNA modifications), but also on the level of chromatin and genome organization. In this review we focus on recent findings that have contributed to our understanding of higher order chromatin structure and genome organization within the nucleus. We highlight new findings on the dynamic positioning of genes relative to each other, as well as to their chromosome territory and the nuclear lamina, and how the position of genes correlates with their transcriptional activity.

Regulation of the histone H4 monomethylase PR-Set7 by CRL4(Cdt2)-mediated PCNA-dependent degradation during DNA damage.

The histone methyltransferase PR-Set7/Set8 is the sole enzyme that catalyzes monomethylation of histone H4 at K20 (H4K20me1). Previous reports document disparate evidence regarding PR-Set7 expression during the cell cycle, the biological relevance of PR-Set7 interaction with PCNA, and its role in the cell. We find that PR-Set7 is indeed undetectable during S phase and instead is detected during late G2, mitosis, and early G1. PR-Set7 is transiently recruited to laser-induced DNA damage sites through its interaction with PCNA, after which 53BP1 is recruited dependent on PR-Set7 catalytic activity. During the DNA damage response, PR-Set7 interaction with PCNA through a specialized "PIP degron" domain targets it for PCNA-coupled CRL4(Cdt2)-dependent proteolysis. PR-Set7 mutant in its "PIP degron" is now detectable during S phase, during which the mutant protein accumulates. Outside the chromatin context, Skp2 promotes PR-Set7 degradation as well. These findings demonstrate a stringent spatiotemporal control of PR-Set7 that is essential for preserving the genomic integrity of mammalian cells.

Nuclear organization and dynamics of 7SK RNA in regulating gene expression.

Noncoding RNAs play important roles in various aspects of gene regulation. We have identified 7SK RNA to be enriched in nuclear speckles or interchromatin granule clusters (IGCs), a subnuclear domain enriched in pre-mRNA processing factors. 7SK RNA, in association with HEXIM 1 and 2, is involved in the inhibition of transcriptional elongation by RNA polymerase II. Inhibition occurs via sequestration of the active P-TEFb kinase complex (CDK 9 and Cyclin T1/T2a/b or K) that is involved in phosphorylating the C-terminal domain of RNA polymerase II. Our results demonstrate that knock-down of 7SK RNA, by specific antisense oligonucleotides, results in the mislocalization of nuclear speckle constituents in a transcription-dependent manner, and the transcriptional up-regulation of a RNA polymerase II transcribed reporter gene locus. Furthermore, 7SK RNA transiently associates with a stably integrated reporter gene locus upon transcriptional down-regulation and its presence correlates with the efficient displacement of P-TEFb constituents from the locus. Our results suggest that 7SK RNA plays a role in modulating the available level of P-TEFb upon transcriptional down-regulation by sequestering its constituents in nuclear speckles.

Chromatin dynamics.

The expression patterns of many protein-coding genes are orchestrated in response to exogenous stimuli, as well as cell-type-specific developmental programs. In recent years, researchers have shown that dynamic chromatin movements and interactions in the nucleus play a crucial role in gene regulation. In this review, we highlight our current understanding of the organization of chromatin in the interphase nucleus and the impact of chromatin dynamics on gene expression. We also discuss the current state of knowledge with regard to the localization of active and inactive genes within the three-dimensional nuclear space. Furthermore, we address recent findings that demonstrate the movements of chromosomal regions and genomic loci in association with changes in transcriptional activity. Finally, we discuss the role of intra- and interchromosomal interactions in the control of coregulated genes.

Transcriptional complexes engaged by apo-estrogen receptor-alpha isoforms have divergent outcomes.

Unliganded (apo-) estrogen receptor alpha (ERalpha, NR3A1) is classically considered as transcriptionally unproductive. Reassessing this paradigm demonstrated that apo-human ERalpha (ERalpha66) and its N-terminally truncated isoform (ERalpha46) are both predominantly nuclear transcription factors that cycle on the endogenous estrogen-responsive pS2 gene promoter in vivo. Importantly, isoform-specific consequences occur in terms of poising the promoter for transcription, as evaluated by determining (i) the engagement of several cofactors and the resulting nucleosomal organization; and (ii) the CpG methylation state of the pS2 promoter. Although transcriptionally unproductive, cycling of apo-ERalpha66 prepares the promoter to respond to ligand, through sequentially targeting chromatin remodeling complexes and general transcription factors. Additionally, apo-ERalpha46 recruits corepressors, following engagement of cofactors identical to those recruited by apo-ERalpha66. Together, these data describe differential activities of ERalpha isoforms. Furthermore, they depict the maintenance of a promoter in a repressed state as a cyclical process that is intrinsically dependent on initial poising of the promoter.

Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter.

Transcriptional activation of a gene involves an orchestrated recruitment of components of the basal transcription machinery and intermediate factors, concomitant with an alteration in local chromatin structure generated by posttranslational modifications of histone tails and nucleosome remodeling. We provide here a comprehensive picture of events resulting in transcriptional activation of a gene, through evaluating the estrogen receptor-alpha (NR3A1) target pS2 gene promoter in MCF-7 cells. This description integrates chromatin remodeling with a kinetic evaluation of cyclical networks of association of 46 transcription factors with the promoter, as determined by chromatin immunoprecipitation assays. We define the concept of a "transcriptional clock" that directs and achieves the sequential and combinatorial assembly of a transcriptionally productive complex on a promoter. Furthermore, the unanticipated findings of key roles for histone deacetylases and nucleosome-remodeling complexes in limiting transcription implies that transcriptional activation is a cyclical process that requires both activating and repressive epigenetic processes.

Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling.

We present an integrated model of hERalpha-mediated transcription where both unliganded and liganded receptors cycle on estrogen-responsive promoters. Using ChIP, FRAP, and biochemical analysis we evaluate hERalpha at several points in these cycles, establishing the ubiquitination status and subnuclear distribution of hERalpha, its mobility, the kinetics of transcriptional activation, and the cyclic recruitment of E3 ligases and the 19S regulatory component of the proteasome. These experiments, together with an evaluation of the inhibition of transcription and proteasome action, demonstrate that proteasome-mediated degradation and hERalpha-mediated transactivation are inherently linked and act to continuously turn over hERalpha on responsive promoters. Cyclic turnover of hERalpha permits continuous responses to changes in the concentration of estradiol.

A dynamic structural model for estrogen receptor-alpha activation by ligands, emphasizing the role of interactions between distant A and E domains.

The functional interplay between different domains of estrogen receptor-alpha (ERalpha, NR3A1) is responsible for the overall properties of the full-length protein. We previously identified an interaction between the N-terminal A and C-terminal domains, which we demonstrate here to repress ligand-independent transactivation and transrepression abilities of ERalpha. Using targeted mutations based on ERalpha structural models, we determine the basis for this interaction that defines a regulatory interplay between ERalpha A domain, corepressors, and ERalpha Helix 12 for binding to the same C-terminal surface. We propose a dynamic model where binding of different ligands influences the A/D-F domain interaction and results in specific functional outcomes. This model gives insights into the dynamic properties of full-length ERalpha and into the structure of unliganded ERalpha.

Formation of an hER alpha-COUP-TFI complex enhances hER alpha AF-1 through Ser118 phosphorylation by MAPK.

The enhancement of the human estrogen receptor alpha (hER alpha, NR3A1) activity by the orphan nuclear receptor COUP-TFI is found to depend on the establishment of a tight hER alpha-COUP-TFI complex. Formation of this complex seems to involve dynamic mechanisms different from those allowing hER alpha homodimerization. Although the hER alpha-COUP-TFI complex is present in all cells tested, the transcriptional cooperation between the two nuclear receptors is restricted to cell lines permissive to hER alpha activation function 1 (AF-1). In these cells, the physical interaction between COUP-TFI and hER alpha increases the affinity of hER alpha for ERK2/p42(MAPK), resulting in an enhanced phosphorylation state of the hER alpha Ser118. hER alpha thus acquires a strengthened AF-1 activity due to its hyperphosphorylation. These data indicate an alternative interaction process between nuclear receptors and demonstrate a novel protein intercommunication pathway that modulates hER alpha AF-1.