VGF is required for recovery after focal stroke.

The high incidence of ischemic stroke worldwide and poor efficacy of neuroprotective drugs has increased the need for novel therapies in stroke recovery. Transcription of the neurosecretory protein VGF (non-acronym) is enhanced following ischemic stroke and proposed to be important for stroke recovery. To determine the requirement for VGF in recovery, we created Vgffl/fl:Nestin-Cre conditional knockout (Vgf cKO) mice and induced a photothrombotic focal ischemic stroke. Naïve Vgf cKO mice had significant less body weight in the absence of gross defects in brain size, cortical lamination, or deficits in locomotor activity compared to wildtype controls. Following a focal stroke, the Vgf cKO mice had greater deficits including impaired recovery of forepaw motor deficits at 2- and 4-weeks post stroke. The increase in deficits occurred in the absence of any difference in lesion size and was accompanied by a striking loss of stroke-induced migration of SVZ-derived immature neurons to the peri-infarct region. Importantly, exogenous adenoviral delivery of VGF (AdVGF) significantly improved recovery in the Vgf cKO mice and was able to rescue the immature neuron migration defect observed. Taken together, our results define a requirement for VGF in post stroke recovery and identify VGF peptides as a potential future therapeutic.

Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors.

DNA sequence and epigenetic information embedded in chromatin must be faithfully duplicated and transmitted to daughter cells during cell division. However, how chromatin assembly and DNA replication are integrated remains unclear. We examined the contribution of the Tousled-like kinases 1 and 2 (TLK1/TLK2) to chromatin assembly and maintenance of replication fork integrity. We show that TLK activity is required for DNA replication and replication-coupled nucleosome assembly and that lack of TLK activity leads to replication fork stalling and the accumulation of single-stranded DNA, a phenotype distinct from ASF1 depletion. Consistent with these results, sustained TLK depletion gives rise to replication-dependent DNA damage and p53-dependent cell cycle arrest in G1. We find that deficient replication-coupled de novo nucleosome assembly renders replication forks unstable and highly dependent on the ATR and CHK1 checkpoint kinases, as well as poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) activity, to avoid collapse. Human cancer data revealed frequent up-regulation of TLK genes and an association with poor patient outcome in multiple types of cancer, and depletion of TLK activity leads to increased replication stress and DNA damage in a panel of cancer cells. Our results reveal a critical role for TLKs in chromatin replication and suppression of replication stress and identify a synergistic lethal relationship with checkpoint signaling and PARP that could be exploited in treatment of a broad range of cancers.

The sub-nucleolar localization of PHF6 defines its role in rDNA transcription and early processing events.

Ribosomal RNA synthesis occurs in the nucleolus and is a tightly regulated process that is targeted in some developmental diseases and hyperactivated in multiple cancers. Subcellular localization and immunoprecipitation coupled mass spectrometry demonstrated that a proportion of plant homeodomain (PHD) finger protein 6 (PHF6) protein is localized within the nucleolus and interacts with proteins involved in ribosomal processing. PHF6 sequence variants cause Börjeson-Forssman-Lehmann syndrome (BFLS, MIM#301900) and are also associated with a female-specific phenotype overlapping with Coffin-Siris syndrome (MIM#135900), T-cell acute lymphoblastic leukemia (MIM#613065), and acute myeloid leukemia (MIM#601626); however, very little is known about its cellular function, including its nucleolar role. HEK 293T cells were treated with RNase A, DNase I, actinomycin D, or 5,6-dichloro-ß-D-ribofuranosylbenzimadole, followed by immunocytochemistry to determine PHF6 sub-nucleolar localization. We observed RNA-dependent localization of PHF6 to the sub-nucleolar fibrillar center (FC) and dense fibrillar component (DFC), at whose interface rRNA transcription occurs. Subsequent ChIP-qPCR analysis revealed strong enrichment of PHF6 across the entire rDNA-coding sequence but not along the intergenic spacer (IGS) region. When rRNA levels were quantified in a PHF6 gain-of-function model, we observed an overall decrease in rRNA transcription, accompanied by a modest increase in repressive promoter-associated RNA (pRNA) and a significant increase in the expression levels of the non-coding IGS36RNA and IGS39RNA transcripts. Collectively, our results demonstrate a role for PHF6 in carefully mediating the overall levels of ribosome biogenesis within a cell.

PHF6 Degrees of Separation: The Multifaceted Roles of a Chromatin Adaptor Protein.

The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the plant homeodomain finger protein 6 (PHF6) gene. Indeed, germline mutations in PHF6 are the cause of the Börjeson-Forssman-Lehmann X-linked intellectual disability syndrome (BFLS), while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis.

Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation.

Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.

PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex.

Mutations in PHF6 are the cause of Börjeson-Forssman-Lehman syndrome (BFLS), an X-linked intellectual disability (XLID) disorder, and both T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). The PHF6 gene encodes a protein with two plant homeodomain (PHD)-like zinc finger domains. As many PHD-like domains function to target chromatin remodelers to post-translationally modified histones, this suggests a role for PHF6 in chromatin regulation. However, PHD domains are usually found in association with a catalytic domain, a feature that is lacking in PHF6. This distinct domain structure and the minimal information on its cellular function prompted us to perform a proteomic screen to identify PHF6 binding partners. We expressed recombinant Flag-tagged PHF6 in HEK 293T cells for coimmunoprecipitation, and analyzed the purified products by mass spectrometry. We identified proteins involved in ribosome biogenesis, RNA splicing, and chromatin regulation, consistent with PHF6 localization to both the nucleoplasm and nucleolus. Notably, PHF6 copurified with multiple constituents of the nucleosome remodeling and deacetylation (NuRD) complex, including CHD4, HDAC1, and RBBP4. We demonstrate that this PHF6-NuRD complex is not present in the nucleolus but is restricted to the nucleoplasm. The association with NuRD represents the first known interaction for PHF6 and implicates it in chromatin regulation.

SCO-ping out the mechanisms underlying the etiology of hydrocephalus.

The heterogeneous nature of congenital hydrocephalus has hampered our understanding of the molecular basis of this common clinical problem. However, disease gene identification and characterization of multiple transgenic mouse models has highlighted the importance of the subcommissural organ (SCO) and the ventricular ependymal (vel) cells. Here, we review how altered development and function of the SCO and vel cells contributes to hydrocephalus.

Characterization of novel isoforms and evaluation of SNF2L/SMARCA1 as a candidate gene for X-linked mental retardation in 12 families linked to Xq25-26.

BACKGROUND: Mutations in genes whose products modify chromatin structure have been recognized as a cause of X-linked mental retardation (XLMR). These genes encode proteins that regulate DNA methylation (MeCP2), modify histones (RSK2 and JARID1C), and remodel nucleosomes through ATP hydrolysis (ATRX). Thus, genes encoding other chromatin modifying proteins should also be considered as disease candidate genes. In this work, we have characterized the SNF2L gene, encoding an ATP-dependent chromatin remodeling protein of the ISWI family, and sequenced the gene in patients from 12 XLMR families linked to Xq25-26. METHODS: We used an in silico and RT-PCR approach to fully characterize specific SNF2L isoforms. Mutation screening was performed in 12 patients from individual families with syndromic or non-syndromic XLMR. We sequenced each of the 25 exons encompassing the entire coding region, complete 5' and 3' untranslated regions, and consensus splice-sites. RESULTS: The SNF2L gene spans 77 kb and is encoded by 25 exons that undergo alternate splicing to generate several distinct transcripts. Specific isoforms are generated through the alternate use of exons 1 and 13, and by the use of alternate donor splice sites within exon 24. Alternate splicing within exon 24 removes a NLS sequence and alters the subcellular distribution of the SNF2L protein. We identified 3 single nucleotide polymorphisms but no mutations in our 12 patients. CONCLUSION: Our results demonstrate that there are numerous splice variants of SNF2L that are expressed in multiple cell types and which alter subcellular localization and function. SNF2L mutations are not a cause of XLMR in our cohort of patients, although we cannot exclude the possibility that regulatory mutations might exist. Nonetheless, SNF2L remains a candidate for XLMR localized to Xq25-26, including the Shashi XLMR syndrome.