Long-term association of a transcription factor with its chromatin binding site can stabilize gene expression and cell fate commitment.

Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl1 is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl1 transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factor Ascl1 can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells

Characteristics and homogeneity of N6-methylation in human genomes.

A novel DNA modification, N-6 methylated deoxyadenosine (m6dA), has recently been discovered in eukaryotic genomes. Despite its low abundance in eukaryotes, m6dA is implicated in human diseases such as cancer. It is therefore important to precisely identify and characterize m6dA in the human genome. Here, we identify m6dA sites at nucleotide level, in different human cells, genome wide. We compare m6dA features between distinct human cells and identify m6dA characteristics in human genomes. Our data demonstrates for the first time that despite low m6dA abundance, the m6dA mark does often occur consistently at the same genomic location within a given human cell type, demonstrating m6dA homogeneity. We further show, for the first time, higher levels of m6dA homogeneity within one chromosome. Most m6dA are found on a single chromosome from a diploid sample, suggesting inheritance. Our transcriptome analysis not only indicates that human genes with m6dA are associated with higher RNA transcript levels but identifies allele-specific gene transcripts showing haplotype-specific m6dA methylation, which are implicated in different biological functions. Our analyses demonstrate the precision and consistency by which the m6dA mark occurs within the human genome, suggesting that m6dA marks are precisely inherited in humans

HIRA dependent H3.3 deposition is required for transcriptional reprogramming following nuclear transfer to Xenopus oocytes.

BACKGROUND: Nuclear reprogramming is potentially important as a route to cell replacement and drug discovery, but little is known about its mechanism. Nuclear transfer to eggs and oocytes attempts to identify the mechanism of this direct route towards reprogramming by natural components. Here we analyze how the reprogramming of nuclei transplanted to Xenopus oocytes exploits the incorporation of the histone variant H3.3. RESULTS: After nuclear transplantation, oocyte-derived H3.3 but not H3.2, is deposited on several regions of the genome including rDNA, major satellite repeats, and the regulatory regions of Oct4. This major H3.3 deposition occurs in absence of DNA replication, and is HIRA-and transcription-dependent. It is necessary for the shift from a somatic- to an oocyte-type of transcription after nuclear transfer. CONCLUSIONS: This study demonstrates that the incorporation of histone H3.3 is an early and necessary step in the direct reprogramming of somatic cell nuclei by oocyte. It suggests that the incorporation of histone H3.3 is necessary during global changes in transcription that accompany changes in cell fate.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Epigenetic stability of repressed states involving the histone variant macroH2A revealed by nuclear transfer to Xenopus oocytes

How various epigenetic mechanisms restrict chromatin plasticity to determine the stability of repressed genes is poorly understood. Nuclear transfer to Xenopus oocytes induces the transcriptional reactivation of previously silenced genes. Recent work suggests that it can be used to analyze the epigenetic stability of repressed states. The notion that the epigenetic state of genes is an important determinant of the efficiency of nuclear reprogramming is supported by the differential reprogramming of given genes from different starting epigenetic configurations. After nuclear transfer, transcription from the inactive X chromosome of post-implantation-derived epiblast stem cells is reactivated. However, the same chromosome is resistant to reactivation when embryonic fibroblasts are used. Here, we discuss different kinds of evidence that link the histone variant macroH2A to the increased stability of repressed states. We focus on developmentally regulated X chromosome inactivation and repression of autosomal pluripotency genes, where macroH2A may help maintain the long-term stability of the differentiated state of somatic cells

Advancing STI care in low/middle-income countries : has STI syndromic management reached its use-by date?

CAPRISA, 2017.Abstract available in pdf

Functional characterization of Vif proteins from HIV-1 infected patients with different APOBEC3G haplotypes.

CAPRISA, 2016.Abstract available in PDF file

Depression onset and its association with community HIV prevalence: A geospatial and panel analyses of nationally representative South African data, 2015–2017

The scaling up of antiretroviral therapy services over the past decades has led to a remarkable reduction in HIV infections and HIV-related deaths in South Africa (SA). While this is a step in the right direction, it brings a new public health challenge into focus, namely psychological challenges associated with such chronic and often stigmatising condition in SA, home to the largest HIV epidemic. Given the current lack of national-level evidence, we investigated the role of the HIV epidemic on depression onset in SA using nationally representative panel data from the South African National Income Dynamics Study (SA-NIDS). Our incident cohort consisted of 13,020 sampled adult participants who were depression-free in Wave 4 (baseline year of 2015). We then measured the risk of depression onset in Wave 5 (year 2017) based on the level of HIV prevalence in the community where study participants resided at baseline. A High-resolution map of HIV spatial heterogeneity (i.e., community HIV prevalence) was generated using ordinary kriging mapping methods from a separate nationally representative data source that corresponded to the investigation period. Geospatial analyses were conducted to identify the spatial structure of HIV and depression onset, and generalised estimating equations (GEE) regression models were fitted to determine the risk of depression onset over time based on community HIV prevalence. Our geospatial analyses indicated that HIV and depression onset prevalence spatially overlapped in the eastern part of the country, particularly in Gauteng, KwaZulu-Natal, Mpumalanga, and Free State province. The GEE regression analyses indicated that individual residency in a community with high HIV prevalence was significantly associated with a higher risk of depression than a low HIV prevalence community (adjusted odds ratio =1.45, 95% CI=1.12-1.48). For the first time, we identified a geospatial overlap between HIV and depression, with a greater risk of depression onset in high HIV prevalence communities, at a national scale in SA. There is a need for place-based policy interventions that prioritise the availability of and access to mental health services in high HIV prevalent SA communities, in an ageing HIV epidemic

Dependence on a variable residue limits the breadth of an HIV MPER neutralizing antibody, despite convergent evolution with broadly neutralizing antibodies

Broadly neutralizing antibodies (bNAbs) that target the membrane-proximal external region (MPER) of HIV gp41 envelope, such as 4E10, VRC42.01 and PGZL1, can neutralize \u3e 80 % of viruses. These three MPER-directed monoclonal antibodies share germline antibody genes (IGHV1 - 69 and IGKV3 - 20) and form a bNAb epitope class. Furthermore, convergent evolution within these two lineages towards a 111.2GW111.3 motif in the CDRH3 is known to enhance neutralization potency. We have previously isolated an MPER neutralizing antibody, CAP206 - CH12, that uses these same germline heavy and light chain genes but lacks breadth (neutralizing only 6 % of heterologous viruses). Longitudinal sequencing of the CAP206-CH12 lineage over three years revealed similar convergent evolution towards 111.2GW111.3 among some lineage members. Mutagenesis of CAP206-CH12 from 111.2GL111.3 to 111.2GW111.3 and the introduction of the double GWGW motif into CAP206-CH12 modestly improved neutralization potency (2.5 3 -fold) but did not reach the levels of potency of VRC42.01, 4E10 or PGZL1. To explore the lack of potency/breadth, viral mutagenesis was performed to map the CAP206-CH12 epitope. This indicated that CAP206-CH12 is dependent on D674, a highly variable residue at the solvent-exposed elbow of MPER. In contrast, VRC42.01, PGZL1 and 4E10 were dependent on highly conserved residues (W672, F673, T676, andW680) facing the hydrophobic patch of the MPER. Therefore, while CAP206-CH12, VRC42.01, PGZL1 and 4E10 share germline genes and show some evidence of convergent evolution, their dependence on different amino acids, which impacts orientation of binding to the MPER, result in differences in breadth and potency. These data have implications for the design of HIV vaccines directed at the MPER epitope

HIV-1 superinfection resembles primary infection.

CAPRISA, 2015.Abstract available in pdf