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SummaryEpigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf+/− tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/− tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.

Expansion of the polymorphic CGG repeats within the 5'-UTR of the FMR1 gene is associated with variable transcriptional regulation of FMR1. Here we report a novel gene, ASFMR1, overlapping the CGG repeat region of FMR1 and transcribed in the antisense orientation. The ASFMR1 transcript is spliced, polyadenylated and exported to the cytoplasm. Similar to FMR1, ASFMR1 is upregulated in individuals with premutation alleles and is not expressed from full mutation alleles. Moreover, it exhibits premutation-specific alternative splicing. Taken together, these observations suggest that in addition to FMR1, ASFMR1 may contribute to the variable phenotypes associated with the CGG repeat expansion.

Facioscapulohumeral Disease (FSHD) is a dominantly inherited progressive myopathy associated with aberrant production of the transcription factor, Double Homeobox Protein 4 (DUX4). The expression of DUX4 depends on an open chromatin conformation of the D4Z4 macrosatellite array and a specific haplotype on chromosome 4. Even when these requirements are met, DUX4 transcripts and protein are only detectable in a subset of cells indicating that additional constraints govern DUX4 production. Since the direction of transcription, along with the production of non-coding antisense transcripts is an important regulatory feature of other macrosatellite repeats, we developed constructs that contain the non-coding region of a single D4Z4 unit flanked by genes that report transcriptional activity in the sense and antisense directions. We found that D4Z4 contains two promoters that initiate sense and antisense transcription within the array, and that antisense transcription predominates. Transcriptional start sites for the antisense transcripts, as well as D4Z4 regions that regulate the balance of sense and antisense transcripts were identified. We show that the choice of transcriptional direction is reversible but not mutually exclusive, since sense and antisense reporter activity was often present in the same cell and simultaneously upregulated during myotube formation. Similarly, levels of endogenous sense and antisense D4Z4 transcripts were upregulated in FSHD myotubes. These studies offer insight into the autonomous distribution of muscle weakness that is characteristic of FSHD.

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5-E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16-32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development.

The pathway(s) by which disuse is transduced into locally mediated osteoclastic resorption remain unknown. We found that both acute disuse (in vivo) and direct hypoxia (in vitro) induced rapid upregulation of OPN expression by osteocytes. Within the context of OPN's role in osteoclast migration and attachment, hypoxia-induced osteocyte OPN expression may serve to mediate disuse-induced bone resorption. Introduction: We have recently reported that disuse induces osteocyte hypoxia. Because hypoxia upregulates osteopontin (OPN) in nonconnective tissue cells, we hypothesized that both disuse and hypoxia would rapidly elevate expression of OPN by osteocytes. Materials and Methods: The response of osteocytes to 24 h of disuse was explored by isolating the left ulna diaphysis of adult male turkeys from loading (n = 5). Cortical osteocytes staining positive for OPN were determined using immunohistochemistry and confocal microscopy. In vitro experiments were performed to determine if OPN expression was altered in MLO-Y4 osteocytes by direct hypoxia (3, 6, 24, and 48 h) or hypoxia (3 and 24 h) followed by 24 h of reoxygenation. A final in vitro experiment explored the potential of protein kinase C (PKC) to regulate hypoxia-induced osteocyte OPN mRNA alterations. Results: We found that 24 h of disuse significantly elevated osteocyte OPN expression in vivo (145% versus intact bones; p = 0.02). We confirmed this finding in vitro, by observing rapid and significant upregulation of OPN protein expression after 24 and 48 h of hypoxia. Whereas 24 h of reoxygenation after 3 h of hypoxia restored normal osteocyte OPN expression levels, 24 h of reoxygenation after 24 h of hypoxia did not mitigate elevated osteocyte OPN expression. Finally, preliminary inhibitor studies suggested that PKC serves as a potent upstream regulator of hypoxia-induced osteocyte OPN expression. Conclusions: Given the documented roles of OPN as a mediator of environmental stress (e.g., hypoxia), an osteoclast chemotaxant, and a modulator of osteoclastic attachment to bone, we speculate that hypoxia-induced osteocyte OPN expression may serve to mediate disuse-induced osteoclastic resorption. Furthermore, it seems that a brief window of time exists in which reoxygenation (as might be achieved by reloading bone) can serve to inhibit this pathway.

Background In mammals, X chromosome genes are present in one copy in males and two in females. To balance the dosage of X-linked gene expression between the sexes, one of the X chromosomes in females is silenced. X inactivation is initiated by upregulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers. The inactivated X chromosome becomes heterochromatic and visits a specific nuclear compartment adjacent to the nucleolus. Results Here, we show a novel role for the lncRNA Firre in anchoring the inactive mouse X chromosome and preserving one of its main epigenetic features, H3K27me3. Similar to Dxz4, Firre is X-linked and expressed from a macrosatellite repeat locus associated with a cluster of CTCF and cohesin binding sites, and is preferentially located adjacent to the nucleolus. CTCF binding present initially in both male and female mouse embryonic stem cells is lost from the active X during development. Knockdown of Firre disrupts perinucleolar targeting and H3K27me3 levels in mouse fibroblasts, demonstrating a role in maintenance of an important epigenetic feature of the inactive X chromosome. No X-linked gene reactivation is seen after Firre knockdown; however, a compensatory increase in the expression of chromatin modifier genes implicated in X silencing is observed. Further experiments in female embryonic stem cells suggest that Firre does not play a role in X inactivation onset. Conclusions The X-linked lncRNA Firre helps to position the inactive X chromosome near the nucleolus and to preserve one of its main epigenetic features. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0618-0) contains supplementary material, which is available to authorized users.