Spatial and topological organization of DNA chains induced by gene co-localization

Transcriptional activity has been shown to relate to the organization of chromosomes in the eukaryotic nucleus and in the bacterial nucleoid. In particular, highly transcribed genes, RNA polymerases and transcription factors gather into discrete spatial foci called transcription factories. However, the mechanisms underlying the formation of these foci and the resulting topological order of the chromosome remain to be elucidated. Here we consider a thermodynamic framework based on a worm-like chain model of chromosomes where sparse designated sites along the DNA are able to interact whenever they are spatially close-by. This is motivated by recurrent evidence that there exists physical interactions between genes that operate together. Three important results come out of this simple framework. First, the resulting formation of transcription foci can be viewed as a micro-phase separation of the interacting sites from the rest of the DNA. In this respect, a thermodynamic analysis suggests transcription factors to be appropriate candidates for mediating the physical interactions between genes. Next, numerical simulations of the polymer reveal a rich variety of phases that are associated with different topological orderings, each providing a way to increase the local concentrations of the interacting sites. Finally, the numerical results show that both one-dimensional clustering and periodic location of the binding sites along the DNA, which have been observed in several organisms, make the spatial co-localization of multiple families of genes particularly efficient.Comment: Figures and Supplementary Material freely available on http://dx.doi.org/10.1371/journal.pcbi.100067

The FERM and PDZ Domain-Containing Protein Tyrosine Phosphatases, PTPN4 and PTPN3, Are Both Dispensable for T Cell Receptor Signal Transduction

PTPN3 and PTPN4 are two closely-related non-receptor protein tyrosine phosphatases (PTP) that, in addition to a PTP domain, contain FERM (Band 4.1, Ezrin, Radixin, and Moesin) and PDZ (PSD-95, Dlg, ZO-1) domains. Both PTP have been implicated as negative-regulators of early signal transduction through the T cell antigen receptor (TCR), acting to dephosphorylate the TCRζ chain, a component of the TCR complex. Previously, we reported upon the production and characterization of PTPN3-deficient mice which show normal TCR signal transduction and T cell function. To address if the lack of a T cell phenotype in PTPN3-deficient mice can be explained by functional redundancy of PTPN3 with PTPN4, we generated PTPN4-deficient and PTPN4/PTPN3 double-deficient mice. As in PTPN3 mutants, T cell development and homeostasis and TCR-induced cytokine synthesis and proliferation were found to be normal in PTPN4-deficient and PTPN4/PTPN3 double-deficient mice. PTPN13 is another FERM and PDZ domain-containing non-receptor PTP that is distantly-related to PTPN3 and PTPN4 and which has been shown to function as a negative-regulator of T helper-1 (Th1) and Th2 differentiation. Therefore, to determine if PTPN13 might compensate for the loss of PTPN3 and PTPN4 in T cells, we generated mice that lack functional forms of all three PTP. T cells from triple-mutant mice developed normally and showed normal cytokine secretion and proliferative responses to TCR stimulation. Furthermore, T cell differentiation along the Th1, Th2 and Th17 lineages was largely unaffected in triple-mutants. We conclude that PTPN3 and PTPN4 are dispensable for TCR signal transduction

Targeted Deficiency of the Transcriptional Activator Hnf1α Alters Subnuclear Positioning of Its Genomic Targets

DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary β-cells and hepatocytes freshly isolated from mice lacking Hnf1α, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3). We show that in Hnf1a−/− cells inactive endogenous Hnf1α-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1α-targets in Hnf1a−/− cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease

NF-Y Dependent Epigenetic Modifications Discriminate between Proliferating and Postmitotic Tissue

The regulation of gene transcription requires posttranslational modifications of histones that, in concert with chromatin remodeling factors, shape the structure of chromatin. It is currently under intense investigation how this structure is modulated, in particular in the context of proliferation and differentiation. Compelling evidence suggests that the transcription factor NF-Y acts as a master regulator of cell cycle progression, activating the transcription of many cell cycle regulatory genes. However, the underlying molecular mechanisms are not yet completely understood. Here we show that NF-Y exerts its effect on transcription through the modulation of the histone “code”. NF-Y colocalizes with nascent RNA, while RNA polymerase II is I phosphorylated on serine 2 of the YSPTSPS repeats within its carboxyterminal domain and histones are carrying modifications that represent activation signals of gene expression (H3K9ac and PAN-H4ac). Comparing postmitotic muscle tissue from normal mice and proliferating muscles from mdx mice, we demonstrate by chromatin immunoprecipitation (ChIP) that NF-Y DNA binding activity correlates with the accumulation of acetylated histones H3 and H4 on promoters of key cell cycle regulatory genes, and with their active transcription. Accordingly, p300 is recruited onto the chromatin of NF-Y target genes in a NF-Y-dependent manner, as demonstrated by Re-ChIP. Conversely, the loss of NF-Y binding correlates with a decrease of acetylated histones, the recruitment of HDAC1, and a repressed heterochromatic state with enrichment of histones carrying modifications known to mediate silencing of gene expression (H3K9me3, H3K27me2 and H4K20me3). As a consequence, NF-Y target genes are downregulated in this context. In conclusion, our data indicate a role of NF-Y in modulating the structure and transcriptional competence of chromatin in vivo and support a model in which NF-Y-dependent histone “code” changes contribute to the proper discrimination between proliferating and postmitotic cells in vivo and in vitro

Pair-Wise Regulation of Convergence and Extension Cell Movements by Four Phosphatases via RhoA

Various signaling pathways regulate shaping of the main body axis during early vertebrate development. Here, we focused on the role of protein-tyrosine phosphatase signaling in convergence and extension cell movements. We identified Ptpn20 as a structural paralogue of PTP-BL and both phosphatases were required for normal gastrulation cell movements. Interestingly, knockdowns of PTP-BL and Ptpn20 evoked similar developmental defects as knockdown of RPTPα and PTPε. Co-knockdown of RPTPα and PTP-BL, but not Ptpn20, had synergistic effects and conversely, PTPε and Ptpn20, but not PTP-BL, cooperated, demonstrating the specificity of our approach. RPTPα and PTPε knockdowns were rescued by constitutively active RhoA, whereas PTP-BL and Ptpn20 knockdowns were rescued by dominant negative RhoA. Consistently, RPTPα and PTP-BL had opposite effects on RhoA activation, both in a PTP-dependent manner. Downstream of the PTPs, we identified NGEF and Arhgap29, regulating RhoA activation and inactivation, respectively, in convergence and extension cell movements. We propose a model in which two phosphatases activate RhoA and two phosphatases inhibit RhoA, resulting in proper cell polarization and normal convergence and extension cell movements

Intergenic Transcription, Cell-Cycle and the Developmentally Regulated Epigenetic Profile of the Human Beta-Globin Locus

Several lines of evidence have established strong links between transcriptional activity and specific post-translation modifications of histones. Here we show using RNA FISH that in erythroid cells, intergenic transcription in the human β-globin locus occurs over a region of greater than 250 kb including several genes in the nearby olfactory receptor gene cluster. This entire region is transcribed during S phase of the cell cycle. However, within this region there are ∼20 kb sub-domains of high intergenic transcription that occurs outside of S phase. These sub-domains are developmentally regulated and enriched with high levels of active modifications primarily to histone H3. The sub-domains correspond to the β-globin locus control region, which is active at all developmental stages in erythroid cells, and the region flanking the developmentally regulated, active globin genes. These results correlate high levels of non-S phase intergenic transcription with domain-wide active histone modifications to histone H3