TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing.

Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins.

Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource.

Actively transcribed genes adopt a unique chromatin environment with characteristic patterns of enrichment. Within gene bodies, H3K36me3 and cytosine DNA methylation are elevated at exons of spliced genes and have been implicated in the regulation of pre-mRNA splicing. H3K36me3 is further responsive to splicing, wherein splicing inhibition led to a redistribution and general reduction over gene bodies. In contrast, little is known of the mechanisms supporting elevated DNA methylation at actively spliced genic locations. Recent evidence associating the de novo DNA methyltransferase Dnmt3b with H3K36me3-rich chromatin raises the possibility that genic DNA methylation is influenced by splicing-associated H3K36me3. Here, we report the generation of an isogenic resource to test the direct impact of splicing on chromatin. A panel of minigenes of varying splicing potential were integrated into a single FRT site for inducible expression. Profiling of H3K36me3 confirmed the established relationship to splicing, wherein levels were directly correlated with splicing efficiency. In contrast, DNA methylation was equivalently detected across the minigene panel, irrespective of splicing and H3K36me3 status. In addition to revealing a degree of independence between genic H3K36me3 and DNA methylation, these findings highlight the generated minigene panel as a flexible platform for the query of splicing-dependent chromatin modifications.

An ectopic CTCF-dependent transcriptional insulator influences the choice of Vß gene segments for VDJ recombination at TCRß locus.

Insulators regulate transcription as they modulate the interactions between enhancers and promoters by organizing the chromatin into distinct domains. To gain better understanding of the nature of chromatin domains defined by insulators, we analyzed the ability of an insulator to interfere in VDJ recombination, a process that is critically dependent on long-range interactions between diverse types of cis-acting DNA elements. A well-established CTCF-dependent transcriptional insulator, H19 imprint control region (H19-ICR), was inserted in the mouse TCRß locus by genetic manipulation. Analysis of the mutant mice demonstrated that the insulator retains its CTCF and position-dependent enhancer-blocking potential in this heterologous context in vivo. Remarkably, the inserted H19-ICR appears to have the ability to modulate cis-DNA interactions between recombination signal sequence elements of the TCRß locus leading to a dramatically altered usage of Vß segments for Vß-to-DßJß recombination in the mutant mice. This reveals a novel ability of CTCF to govern long range cis-DNA interactions other than enhancer-promoter interactions and suggests that CTCF-dependent insulators may play a diverse and complex role in genome organization beyond transcriptional control. Our functional analysis of mutated TCRß locus supports the emerging role of CTCF in governing VDJ recombination.

Developmental Lead Exposure and Prenatal Stress Result in Sex-Specific Reprograming of Adult Stress Physiology and Epigenetic Profiles in Brain.

Developmental exposure to lead (Pb) and prenatal stress (PS) both impair cognition, which could derive from their joint targeting of the hypothalamic-pituitary-adrenal axis and the brain mesocorticolimbic (MESO) system, including frontal cortex (FC) and hippocampus (HIPP). Glucocorticoids modulate both FC and HIPP function and associated mediation of cognitive and other behavioral functions. This study sought to determine whether developmental Pb ± PS exposures altered glucocorticoid-related epigenetic profiles in brain MESO regions in offspring of female mice exposed to 0 or 100 ppm Pb acetate drinking water from 2 mos prior to breeding until weaning, with half further exposed to prenatal restraint stress from gestational day 11-18. Overall, changes in females occured in response to Pb exposure. In males, however, Pb-induced neurotoxicity was modulated by PS. Changes in serum corticosterone levels were seen in males, while glucocorticoid receptor changes were seen in both sexes. In contrast, both Pb and PS broadly impacted brain DNA methyltransferases and binding proteins, particularly DNMT1, DNMT3a and methyl-CpG-binding protein 2, with patterns that differed by sex and brain regions. Specifically, in males, effects on FC epigenetic modifiers were primarily influenced by Pb, whereas extensive changes in HIPP were produced by PS. In females, Pb exposure and not PS primarily altered epigenetic modifiers in both FC and HIPP. Collectively, these findings indicate that epigenetic mechanisms may underlie associated neurotoxicity of Pb and of PS, particularly associated cognitive deficits. However, mechanisms by which this may occur will be different in males versus females.

Influence of a CTCF-Dependent Insulator on Multiple Aspects of Enhancer-Mediated Chromatin Organization.

Developmental stage-specific enhancer-promoter-insulator interactions regulate the chromatin configuration necessary for transcription at various loci and additionally for VDJ recombination at antigen receptor loci that encode immunoglobulins and T-cell receptors. To investigate these regulatory interactions, we analyzed the epigenetic landscape of the murine T-cell receptor ß (TCRß) locus in the presence and absence of an ectopic CTCF-dependent enhancer-blocking insulator, H19-ICR, in genetically manipulated mice. Our analysis demonstrated the ability of the H19-ICR insulator to restrict several aspects of enhancer-based chromatin alterations that are observed during activation of the TCRß locus for transcription and recombination. The H19-ICR insulator abrogated enhancer-promoter contact-dependent chromatin alterations and additionally prevented Eß-mediated histone modifications that have been suggested to be independent of enhancer-promoter interaction. Observed enhancer-promoter-insulator interactions, in conjunction with the chromatin structure of the Eß-regulated domain at the nucleosomal level, provide useful insights regarding the activity of the regulatory elements in addition to supporting the accessibility hypothesis of VDJ recombination. Analysis of H19-ICR in the heterologous context of the developmentally regulated TCRß locus suggests that different mechanisms proposed for CTCF-dependent insulator action might be manifested simultaneously or selectively depending on the genomic context and the nature of enhancer activity being curtailed.

Serotype profile and molecular characterization of antimicrobial resistance in non-typhoidal Salmonella isolated from gastroenteritis cases over nine years.

Resistance to third-generation cephalosporins in non-typhoidal Salmonella (NTS) is emerging worldwide. We report the occurrence of extended-spectrum beta-lactamase (ESBL) phenotypes in 53.4 % of NTS isolated over a period of nine years from gastroenteritis cases. ESBL and AmpC co-production was observed in 21 % of the isolates. Occurrence of blaCTX-M-15 and blaCMY-2 resistance genes was observed in 11.6 % and 37 % of the isolates respectively. Overall, Salmonella enterica serovar Senftenberg was the predominant serovar carrying blaCTX-M-15 and blaCMY-2 resistance genes. We report for the first time from India, one isolate each of S. enterica serovar Thompson, S. enterica serovar Infantis and S. enterica serovar Newport, carrying the blaCTX-M-15 gene. We also report for the first time from India, a case of gastroenteritis due to S. enterica serovar Thompson.