Functional interplay between MyoD and CTCF in regulating long-range chromatin interactions during differentiation.

Higher-order chromatin structures appear to be dynamically arranged during development and differentiation. However, the molecular mechanism underlying their maintenance or disruption and their functional relevance to gene regulation are poorly understood. We recently described a dynamic long-range chromatin interaction between the gene promoter of the cdk inhibitor p57(kip2) (also known as Cdkn1c) and the imprinting control region KvDMR1 in muscle cells. Here, we show that CTCF, the best characterized organizer of long-range chromatin interactions, binds to both the p57(kip2) promoter and KvDMR1 and is necessary for the maintenance of their physical contact. Moreover, we show that CTCF-mediated looping is required to prevent p57(kip2) expression before differentiation. Finally, we provide evidence that the induction of p57(kip2) during myogenesis involves the physical interaction of the muscle-regulatory factor MyoD with CTCF at KvDMR1, the displacement of the cohesin complex subunit Rad21 and the destabilization of the chromatin loop. The finding that MyoD affects chromatin looping at CTCF-binding sites represents the first evidence that a differentiation factor regulates chromatin-loop dynamics and provides a useful paradigm for gaining insights into the developmental regulation of long-range chromatin contacts.

A highly specific q-RT-PCR assay to address the relevance of the JAK2WT and JAK2V617F expression levels and control genes in Ph-negative myeloproliferative neoplasms.

In Ph- myeloproliferative neoplasms, the quantification of the JAK2V617F transcripts may provide some advantages over the DNA allele burden determination. We developed a q-RT-PCR to assess the JAK2WT and JAK2V617F mRNA expression in 105 cases (23 donors, 13 secondary polycythemia, 22 polycythemia vera (PV), 38 essential thrombocythemia (ET), and 9 primary myelofibrosis (PMF)). Compared with the standard allele-specific oligonucleotide (ASO)-PCR technique, our assay showed a 100 % concordance rate detecting the JAK2V617F mutation in 22/22 PV (100 %), 29/38 (76.3 %) ET, and 5/9 (55.5 %) PMF cases, respectively. The sensitivity of the assay was 0.01 %. Comparing DNA and RNA samples, we found that the JAK2V617F mutational ratios were significantly higher at the RNA level both in PV (p = 0.005) and ET (p = 0.001) samples. In PV patients, JAK2WT expression levels positively correlated with the platelets (PLTs) (p = 0.003) whereas a trend to negative correlation was observed with the Hb levels (p = 0.051). JAK2V617F-positive cases showed the lowest JAK2WT and ABL1 mRNA expression levels. In all the samples, the expression pattern of beta-glucoronidase (GUSB) was more homogeneous than that of ABL1 or ß2 microglobulin (B2M). Using GUSB as normalizator gene, a significant increase of the JAK2V617F mRNA levels was seen in two ET patients at time of progression to PV. In conclusion, the proposed q-RT-PCR is a sensitive and accurate method to quantify the JAK2 mutational status that can also show clinical correlations suggesting the impact of the residual amount of the JAK2WT allele on the Ph- MPN disease phenotype. Our observations also preclude the use of ABL1 as a housekeeping gene for these neoplasms.

MyoD regulates p57kip2 expression by interacting with a distant cis-element and modifying a higher order chromatin structure.

The bHLH transcription factor MyoD, the prototypical master regulator of differentiation, directs a complex program of gene expression during skeletal myogenesis. The up-regulation of the cdk inhibitor p57kip2 plays a critical role in coordinating differentiation and growth arrest during muscle development, as well as in other tissues. p57kip2 displays a highly specific expression pattern and is subject to a complex epigenetic control driving the imprinting of the paternal allele. However, the regulatory mechanisms governing its expression during development are still poorly understood. We have identified an unexpected mechanism by which MyoD regulates p57kip2 transcription in differentiating muscle cells. We show that the induction of p57kip2 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a chromatin loop involving p57kip2 promoter. We also show that differentiation-dependent regulation of p57kip2, while involving a region implicated in the imprinting process, is distinct and hierarchically subordinated to the imprinting control. These findings highlight a novel mechanism, involving the modification of higher order chromatin structures, by which MyoD regulates gene expression. Our results also suggest that chromatin folding mediated by KvDMR1 could account for the highly restricted expression of p57kip2 during development and, possibly, for its aberrant silencing in some pathologies.

Mutation in the VP1-LDV motif of the murine polyomavirus affects viral infectivity and conditions virus tissue tropism in vivo.

The first contact of a virus with the host cell surface and further entry are important steps for a successful outcome of the infection process and for the virus-associated pathogenicity. We have previously shown that the entry of the murine Polyomavirus (Py) into fibroblasts is a multi-step process involving, at least, the attachment to primary sialic acids (SA)-containing cell receptors followed by post-binding interaction with secondary receptors, such as the alpha4beta1 integrin, likely through the VP1-LDV motif. Here we report on the functional role of the VP1-LDV motif in Py infectivity and in vivo virus tissue tropism. For this purpose, we have characterized a recombinant virus mutant, PyLNV, harboring a single aa substitution in this motif (D138N). Although not critical for virus viability, the D138N substitution abrogates the post-attachment Py-alpha4beta1 interaction, rendering the PyLNV mutant virus twofold less infectious than the Py wild-type (Wt) in alpha4beta1-positive fibroblasts. To study the putative role of the VP1-LDV motif in vivo, newborn C57BL/6 mice were inoculated with PyWt or PyLNV and, after six days, organs were analyzed for the presence of viral DNA. Intriguingly, PyLNV showed an altered spectrum of in vivo replication compared with PyWt, particularly in the skin and in the kidney. The implication of Py-alpha4beta1 integrin interaction in conditioning tissue-specificity of virus replication is discussed.

Review: Environmental impact on ocular surface disorders: Possible epigenetic mechanism modulation and potential biomarkers.

Throughout life, external and the internal environments interact in normal development and tissue homeostasis as well as in onset of disease. Epigenetic modifications occur in response to environmental changes and play a fundamental role in controlling gene expression without modification of the DNA base sequence. Aging, inflammation, drugs, infections and ultraviolet exposure may have profound effects on epigenetic modifications and trigger susceptibility to diseases. Increasing evidence has demonstrated that epigenetic mechanisms play a key role in regulating the physiopathology of the ocular surface. The evaluation of epigenetic factors in ocular disease would lead to further investigation regarding the potential use of therapeutic targets and diagnostic biomarkers. This review examines specific epigenetic and biochemical mechanisms that may occur in the ocular surface microenvironment. Potential correlation between epigenetic factors and targets should be considered in future approaches to prevention and/or treatment of specific ocular surface disorders.

Exploring Serum Levels of Brain Derived Neurotrophic Factor and Nerve Growth Factor Across Glaucoma Stages.

PURPOSE: To investigate the serum levels of Brain Derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) in patients affected by primary open angle glaucoma with a wide spectrum of disease severity compared to healthy controls and to explore their relationship with morphological and functional glaucoma parameters. MATERIALS AND METHODS: 45 patients affected by glaucoma at different stages and 15 age-matched healthy control subjects underwent visual field testing, peripapillary retinal nerve fibre layer thickness measurement using Spectral Domain Optical Coherence Tomography and blood collection for both neurotrophins detection by Enzyme-Linked Immunosorbent Assay. Statistical analysis and association between biostrumental and biochemical data were investigated. RESULTS: Serum levels of BDNF in glaucoma patients were significantly lower than those measured in healthy controls (261.2±75.0 pg/ml vs 313.6±79.6 pg/ml, p = 0.03). Subgroups analysis showed that serum levels of BDNF were significantly lower in early (253.8±40.7 pg/ml, p = 0.019) and moderate glaucoma (231.3±54.3 pg/ml, p = 0.04) but not in advanced glaucoma (296.2±103.1 pg/ml, p = 0.06) compared to healthy controls. Serum levels of NGF in glaucoma patients were significantly lower than those measured in the healthy controls (4.1±1 pg/mL vs 5.5±1.2 pg/mL, p = 0.01). Subgroups analysis showed that serum levels of NGF were significantly lower in early (3.5±0.9 pg/mL, p = 0.0008) and moderate glaucoma (3.8±0.7 pg/ml, p<0.0001) but not in advanced glaucoma (5.0±0.7 pg/ml, p = 0.32) compared to healthy controls. BDNF serum levels were not related to age, visual field mean deviation or retinal nerve fibre layer thickness either in glaucoma or in controls while NGF levels were significantly related to visual field mean deviation in the glaucoma group (r2 = 0.26, p = 0.004). CONCLUSIONS: BDNF and NGF serum levels are reduced in the early and moderate glaucoma stages, suggesting the possibility that both factors could be further investigated as potential circulating biomarkers for the early detection of glaucoma.

Regulation of p57(KIP2) during muscle differentiation: role of Egr1, Sp1 and DNA hypomethylation.

The cdk inhibitor p57(kip2) plays a critical role in many differentiation processes by performing not only redundant but also specific functions. Compared to other cdk inhibitors, p57(kip2) shows a more restricted expression pattern during development and in adult tissues. We have previously reported that in muscle cells, p57(kip2) is induced by the myogenic factor MyoD through an indirect mechanism involving p73 proteins as intermediaries. We have also reported that p57(kip2) shows a differential responsiveness to MyoD-dependent regulation in different cell types. In this work we have further investigated the molecular mechanism by which MyoD activates p57 promoter. We show that the minimal promoter element able to confer MyoD responsiveness contains multiple Sp1 and Egr1 recognition sites and that both transcription factors are necessary for the increase in p57 RNA. We also suggest that the role of MyoD-induced p73 consists in promoting the binding of Sp1 to p57(kip2) promoter. Moreover, we show that Egr1 and Sp1 are concomitantly recruited to p57 promoter in vivo only in differentiation conditions and only in responsive cells. Bisulfite sequencing suggested a functional link between the methylation status and the differential activity of p57 promoter, both during differentiation and in distinct cell types. These results, which highlight the involvement of epigenetic factors in the regulation of p57 expression in muscle cells, could be of general relevance to explain its tissue and cell type restriction during development.