Nuclear receptor-enhanced transcription requires motor- and LSD1-dependent gene networking in interchromatin granules.

While the transcriptional machinery has been extensively dissected at the molecular level, little is known about regulation of chromosomal organization in the three-dimensional space of the nucleus to achieve integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories requiring nuclear actin/myosin-I transport machinery, dynein light chain 1 (DLC1), and a specific subset of transcriptional coactivators and chromatin remodeling complexes. We establish a requirement for the histone lysine demethylase, LSD1, in directing specific interchromosomal interaction loci to distinct interchromatin granules, long thought to be "storage" sites for splicing machinery, and demonstrate that these three-dimensional motor-dependent interactions are required to achieve enhanced transcription of specific estrogen-receptor target genes. These findings reveal roles for the modulation of nuclear architecture in orchestrating regulated gene-expression programs in the mammalian nucleus.

Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation.

The functional importance of gene enhancers in regulated gene expression is well established. In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells, bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs). However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17ß-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer-promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer-promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.

Enhancing nuclear receptor-induced transcription requires nuclear motor and LSD1-dependent gene networking in interchromatin granules.

Although the role of liganded nuclear receptors in mediating coactivator/corepressor exchange is well-established, little is known about the potential regulation of chromosomal organization in the 3-dimensional space of the nucleus in achieving integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among specific subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories, which depends on the actions of nuclear actin/myosin-I machinery and dynein light chain 1. The histone lysine demethylase, LSD1, is required for these ligand-induced interactive loci to associate with distinct interchromatin granules, long thought to serve as "storage" sites for the splicing machinery, some critical transcription elongation factors, and various chromatin remodeling complexes. We demonstrate that this 2-step nuclear rearrangement is essential for achieving enhanced, coordinated transcription of nuclear receptor target genes.

A sensitive array-based assay for identifying multiple TMPRSS2:ERG fusion gene variants.

Studies of gene fusions in solid tumors are not as extensive as in hematological malignancies due to several technical and analytical problems associated with tumor heterogeneity. Nevertheless, there is a growing interest in the role of fusion genes in common epithelial tumors after the discovery of recurrent TMPRSS2:ETS fusions in prostate cancer. Among all of the reported fusion partners in the ETS gene family, TMPRSS2:ERG is the most prevalent one. Here, we present a simple and sensitive microarray-based assay that is able to simultaneously determine multiple fusion variants with a single RT-PCR in impure RNA specimens. The assay detected TMPRSS2:ERG fusion transcripts with a detection sensitivity of <10 cells in the presence of more than 3000 times excess normal RNA, and in primary prostate tumors having no >1% of cancer cells. The ability to detect multiple transcript variants in a single assay is critically dependent on both the primer and probe designs. The assay should facilitate clinical and basic studies for fusion gene screening in clinical specimens, as it can be readily adapted to include multiple gene loci.

Hpr (ScoC) and the phosphorelay couple cell cycle and sporulation in Bacillus subtilis.

Bacillus subtilis sporulation is a developmental process that culminates in the formation of a highly resistant and persistent endospore. Inhibiting DNA synthesis prior to the completion of the final round of DNA replication blocks sporulation at an early stage. Conditions that prevent compartmentalization of gene expression, i.e. inhibition of asymmetric septum formation or chromosome partitioning, also block sporulation at an early stage. Multiple mechanisms including a RecA-dependent, a RecA-independent, and the soj-spo0J operon have been implicated in signal transduction, connecting DNA replication and chromosome partitioning to the onset of sporulation in B. subtilis. We suggest that a single mechanism involving Hpr (ScoC) and Sda couple cell cycle signaling to sporulation initiation. We show that transcription of phosphorelay sensory chain genes is adversely affected by post-exponential perturbation of the cell cycle. DNA replication arrest by chemical treatments, such as hydroxyphenylazouracil, hydroxyurea, nalidixic acid, and through genetic means using dnaA1ts and dnaB19ts temperature-sensitive mutants caused substantial down-regulation of spo0F and kinA expression and elevated the expression of spo0A and spo0H (sigH). Despite the elevation in spo0A expression, Spo0A approximately P-dependent sinI expression was substantially down-regulated indicating that in vivo Spo0A approximately P levels may be diminished. Similar alterations in gene expression patterns were observed in an ftsA279ts mutant background, indicating that cytokinesis and sporulation may also be coupled by a similar mechanism. Loss of function mutation in hpr (scoC) restored sporulation in a dnaA1ts mutant, blocked the DNA replication arrest induction of spo0A expression and restored expression of spo0F, kinA and sinI. Moreover, hpr expression was up-regulated in response to DNA replication arrest. The increase in hpr expression required Sda. These results suggest a role for Hpr (ScoC) in mediating the coupling of cell cycle events to the onset of sporulation.

Nuclear organization in the 3D space of the nucleus - cause or consequence?

Recent evidence suggests that dynamic three-dimensional genomic interactions in the nucleus exert critical roles in regulated gene expression. Here, we review a series of recent paradigm-shifting experiments that highlight the existence of specific gene networks within the self-organizing space of the nucleus. These gene networks, evidenced by long-range intrachromosomal and interchromosomal interactions, can be considered as the cause or consequence of regulatory biological programs. Changes in nuclear architecture are a hallmark of laminopathies and likely potentiate genome rearrangements critical for tumor progression, in addition to potential vital contribution of noncoding RNAs and DNA repeats. It is virtually certain that we will witness an ever-increasing rate of discoveries that uncover new roles of nuclear architecture in transcription, DNA damage/repair, aging, and disease.

Developmentally regulated activation of a SINE B2 repeat as a domain boundary in organogenesis.

The temporal and spatial regulation of gene expression in mammalian development is linked to the establishment of functional chromatin domains. Here, we report that tissue-specific transcription of a retrotransposon repeat in the murine growth hormone locus is required for gene activation. This repeat serves as a boundary to block the influence of repressive chromatin modifications. The repeat element is able to generate short, overlapping Pol II-and Pol III-driven transcripts, both of which are necessary and sufficient to enable a restructuring of the regulated locus into nuclear compartments. These data suggest that transcription of interspersed repetitive sequences may represent a developmental strategy for the establishment of functionally distinct domains within the mammalian genome to control gene activation.

ScoC mediates catabolite repression of sporulation in Bacillus subtilis.

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and sigmaH in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucose.