A Genome-Wide Screen for Genetic Variants That Modify the Recruitment of REST to Its Target Genes

Increasing numbers of human diseases are being linked to genetic variants, but our understanding of the mechanistic links leading from DNA sequence to disease phenotype is limited. The majority of disease-causing nucleotide variants fall within the non-protein-coding portion of the genome, making it likely that they act by altering gene regulatory sequences. We hypothesised that SNPs within the binding sites of the transcriptional repressor REST alter the degree of repression of target genes. Given that changes in the effective concentration of REST contribute to several pathologies—various cancers, Huntington's disease, cardiac hypertrophy, vascular smooth muscle proliferation—these SNPs should alter disease-susceptibility in carriers. We devised a strategy to identify SNPs that affect the recruitment of REST to target genes through the alteration of its DNA recognition element, the RE1. A multi-step screen combining genetic, genomic, and experimental filters yielded 56 polymorphic RE1 sequences with robust and statistically significant differences of affinity between alleles. These SNPs have a considerable effect on the the functional recruitment of REST to DNA in a range of in vitro, reporter gene, and in vivo analyses. Furthermore, we observe allele-specific biases in deeply sequenced chromatin immunoprecipitation data, consistent with predicted differenes in RE1 affinity. Amongst the targets of polymorphic RE1 elements are important disease genes including NPPA, PTPRT, and CDH4. Thus, considerable genetic variation exists in the DNA motifs that connect gene regulatory networks. Recently available ChIP–seq data allow the annotation of human genetic polymorphisms with regulatory information to generate prior hypotheses about their disease-causing mechanism

The neurogenic potential of astrocytes is regulated by inflammatory signals

Although the adult brain contains neural stem cells (NSCs) that generate new neurons throughout life, these astrocyte-like populations are restricted to two discrete niches. Despite their terminally differentiated phenotype, adult parenchymal astrocytes can re-acquire NSC-like characteristics following injury, and as such, these 'reactive' astrocytes offer an alternative source of cells for central nervous system (CNS) repair following injury or disease. At present, the mechanisms that regulate the potential of different types of astrocytes are poorly understood. We used in vitro and ex vivo astrocytes to identify candidate pathways important for regulation of astrocyte potential. Using in vitro neural progenitor cell (NPC)-derived astrocytes, we found that exposure of more lineage-restricted astrocytes to either tumor necrosis factor alpha (TNF-α) (via nuclear factor-κB (NFκB)) or the bone morphogenetic protein (BMP) inhibitor, noggin, led to re-acquisition of NPC properties accompanied by transcriptomic and epigenetic changes consistent with a more neurogenic, NPC-like state. Comparative analyses of microarray data from in vitro-derived and ex vivo postnatal parenchymal astrocytes identified several common pathways and upstream regulators associated with inflammation (including transforming growth factor (TGF)-β1 and peroxisome proliferator-activated receptor gamma (PPARγ)) and cell cycle control (including TP53) as candidate regulators of astrocyte phenotype and potential. We propose that inflammatory signalling may control the normal, progressive restriction in potential of differentiating astrocytes as well as under reactive conditions and represent future targets for therapies to harness the latent neurogenic capacity of parenchymal astrocytes

Cultural respect in general practice : a cluster randomised controlled trial

Objective: To examine whether the Ways of Thinking and Ways of Doing (WoTWoD) cultural respect framework improves clinically appropriate anticipatory care in general practice and the cultural respect levels of medical practice staff. Design: Mixed methods, cluster randomised controlled trial with a participatory action research approach. Setting, participants: Fifty-six general practices in Sydney and Melbourne, 2014–2017. Intervention: WoTWoD encompasses a toolkit (ten scenarios illustrating cross-cultural behaviour in clinical practice), one half-day workshop, cultural mentor support for practices, and a local care partnership between participating Medicare locals/primary health networks and local Aboriginal Community Controlled Health Services for guiding the program and facilitating community engagement. The intervention lasted 12 months at each practice. Major outcomes: Rates of claims for MBS item 715 (health assessment for Aboriginal and Torres Strait Islander People) and recording of chronic disease risk factors; changes in cultural quotient (CQ) scores of practice staff. Results: Complete results were available for 28 intervention (135 GPs, 807 Indigenous patients) and 25 control practices (210 GPs, 1554 Indigenous patients). 12-Month rates of MBS item 715 claims and recording of risk factors for the two groups were not statistically significantly different, nor were mean changes in CQ scores, regardless of staff category and practice attributes. Conclusion: The WoTWoD program did not increase the rate of Indigenous health checks or improve cultural respect scores in general practice. Conceptual, methodologic, and contextual factors that influence cultural mentorship, culturally respectful clinical practice, and Indigenous health care require further investigation. Trial registration: Australia New Zealand Clinical Trials Registry ACTRN12614000797673

Cultural respect in general practice : a mixed method cluster randomised controlled trial

Context: Australia is struggling to address the disparity in health of Indigenous Australians. The Ways of Thinking and Ways of Doing (WoTWoD) cultural respect intervention comprises a toolkit, one half-day workshop, cultural mentor support for practices, and a local care partnership between local Primary Health Networks and Aboriginal Community Controlled Health Services to guide the program and facilitate community engagement. Objective: To examine whether WoTWoD improves clinically appropriate anticipatory care in general practice and cultural respect displayed by medical practice staff as measured by rates of claims for MBS item 715 (health assessment for Indigenous Australians), recording of chronic disease risk factors; and changes in cultural quotient (CQ) scores of practice staff. Design: Mixed methods, cluster randomised controlled trial with a participatory action research approach. Setting/Participants: Fifty-six general practices in Sydney and Melbourne during 2014–2017. Findings: Complete data were available for 28 intervention (135 GPs, 807 Indigenous patients) and 25 control practices (210 GPs, 1554 Indigenous patients). Participants reported positive experiences. However, 12-month rates of MBS item 715 claims and recording of risk factors for the two groups were not statistically significantly different, nor were mean changes in CQ scores, regardless of staff category and practice attributes. Implication(s) for practice: While it was well-received, the WoTWoD program did not increase the rate of Indigenous health checks or improve cultural respect in general practice. Conceptual, methodologic, and contextual factors that influence cultural mentorship, culturally respectful clinical practice, and Indigenous health care require further investigation

Repressor Element 1 Silencing Transcription Factor Couples Loss of Pluripotency with Neural Induction and Neural Differentiation

Neural differentiation of embryonic stem cells (ESCs) requires coordinated repression of the pluripotency regulatory program and reciprocal activation of the neurogenic regulatory program. Upon neural induction, ESCs rapidly repress expression of pluripotency genes followed by staged activation of neural progenitor and differentiated neuronal and glial genes. The transcriptional factors that underlie maintenance of pluripotency are partially characterized whereas those underlying neural induction are much less explored, and the factors that coordinate these two developmental programs are completely unknown. One transcription factor, REST (repressor element 1 silencing transcription factor), has been linked with terminal differentiation of neural progenitors and more recently, and controversially, with control of pluripotency. Here, we show that in the absence of REST, coordination of pluripotency and neural induction is lost and there is a resultant delay in repression of pluripotency genes and a precocious activation of both neural progenitor and differentiated neuronal and glial genes. Furthermore, we show that REST is not required for production of radial glia‐like progenitors but is required for their subsequent maintenance and differentiation into neurons, oligodendrocytes, and astrocytes. We propose that REST acts as a regulatory hub that coordinates timely repression of pluripotency with neural induction and neural differentiation

An epigenetic signature of developmental potential in neural stem cells and early neurons.

A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons. Stem Cells 2013;31:1868-1880

SNPs may increase or decrease affinity of RE1s.

<p>Examples of SNPs that decrease (A) and increase (B) the affinity of an RE1 sequence. On the left are diagrams of the genomic location of polymorphic RE1s, their target genes and the REST ChIPseq read density taken from ENCODE data. On the right is corresponding quantitative EMSA data. In (A), the well-studied RE1 that lies within the 3′ UTR of the <i>NPPA</i> gene contains the SNP rs12565 that strongly decreases its affinity for REST. In (B), the RE1 lying distally upstream of the <i>CDH4</i> gene contains the SNP rs6093022 that strongly increases its affinity. (C) The SNP rs1040480 within an intron of <i>PTPRT</i> reduces the affinity of REST. B = Bound complex of REST with probe; U = Unbound probe; D = Degradation product. The latter band represents a fraction of purified REST protein that is partially degraded, as was observed previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002624#pgen.1002624-Johnson4" target="_blank">[20]</a>.</p

Electrophoretic mobility shift assay to measure affinity differences between RE1 alleles.

<p>To measure RE1 affinity in vitro, we employed a competition EMSA method. We tested the ability of unlabelled competitor sequences to compete for REST binding with a fluorescently labelled DNA probe. (A) Various control oligonucleotides were used to validate the sensitivity and selectivity of the comparative EMSA assay. The Ideal RE1 motif is a high affinity synthetic sequence we used previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002624#pgen.1002624-Johnson4" target="_blank">[20]</a>. By swapping two highly conserved dinucleotides in the sequence, the affinity of the Ideal RE1 can be completely abolished (Mutated RE1). We also designed four pairs of RE1 alleles (N1-4), where SNPs lie outside the RE1 half sites, and thus would not be expected to alter binding affinity. (B) The results of control EMSAs are shown, where replicate EMSA gels have been quantitated and plotted. The data are displayed in units of Fraction Bound (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002624#s4" target="_blank">Materials and Methods</a>), where a low Fraction Bound value indicates high binding affinity, and vice versa. Example raw data are shown in panels: (C) Ideal/Mutated RE1s and (D) N1 RE1s. (E) Summary results of EMSA for all pRE1s in this study. The y-axis plots the difference in Fraction Bound between Major and Minor alleles, where the arrow begins at the value for Major, and ends at Minor. All RE1s are ranked by their change in Fraction Bound.</p

Polymorphic RE1s where the minor allele has increased affinity.

<p>FB: Change in Fraction Bound value, FB−FB; Frequency indicates Hapmap populations where the Minor SNP allele occurs at 5% (ND-Not determined, -5% in all populations)(Note: genotype data come from Hapmap, except for genotyping carried out on Hapmap CEU set in this study, denoted CEU*); Dist: Distance from RE1 to gene transcriptional start site (negative indicate upstream). Known REST target genes are underlined - see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002624#pgen.1002624.s005" target="_blank">File S1</a> for more information.</p

Experimental pipeline to discover SNPs that affect gene repression by REST.

<p>(A) The structure of the RE1 motif, illustrating its two strongly constrained half sites and weakly constrained spacer and 3′ regions. The spacer region may have “canonical” size of two nucleotides, or other “non-canonical” sizes. (B) Cartoon illustrating the hypothetical effect of a SNP in an RE1 element. In the upper panel, the Major (ie more frequent) allele contains a high-affinity RE1 sequence that strongly recruits REST, resulting in target gene repression. The presence of the SNP reduces REST binding affinity, and results in an increase in target gene transcription. (C) The flowchart illustrates the pipeline employed in this study to discover pRE1 SNPs.</p