Son C. Nguyen

Developmental Profile of H19 Differentially Methylated Domain (DMD) Deletion Alleles Reveals Multiple Roles of the DMD in Regulating Allelic Expression and DNA Methylation at the Imprinted H19/Igf2 Locus

ABSTRACT The differentially methylated domain (DMD) of the mouse H19 gene is a methylation-sensitive insulator that blocks access of the Igf2 gene to shared enhancers on the maternal allele and inactivates H19 expression on the methylated paternal allele. By analyzing H19 DMD deletion alleles H19ΔDMD and H19Δ3.8kb-5′H19 in pre- and postimplantation embryos, we show that the DMD exhibits positive transcriptional activity and is required for H19 expression in blastocysts and full activation of H19 during subsequent development. We also show that the DMD is required to establish Igf2 imprinting by blocking access to shared enhancers when Igf2 monoallelic expression is initiated in postimplantation embryos and that the single remaining CTCF site of the H19ΔDMD allele is unable to provide this function. Furthermore, our data demonstrate that sequence outside of the DMD can attract some paternal-allele-specific CpG methylation 5′ of H19 in preimplantation embryos, although this methylation is not maintained during postimplantation in the absence of the DMD. Finally, we report a conditional allele floxing the 1.6-kb sequence deleted from the H19ΔDMD allele and demonstrate that the DMD is required to maintain repression of the maternal Igf2 allele and the full activity of the paternal Igf2 allele in neonatal liver.

HIV-specific CD8 + T cells exhibit reduced and differentially regulated Cytolytic activity in Lymphoid tissue

SUMMARY Elimination of lymphoid tissue reservoirs is a key component of HIV eradication strategies. CD8+ T cells play a critical role in control of HIV, but their functional attributes in lymph nodes (LNs) remain unclear. Here, we show that memory, follicular CXCR5+, and HIV-specific CD8+ T cells from LNs do not manifest the properties of cytolytic CD8+ T cells. While the frequency of follicular CXCR5+ CD8+ T cells was strongly inversely associated with peripheral viremia, this association was not dependent on cytolytic CXCR5+ CD8+ T cells. Moreover, the poor cytolytic activity of LN CD8+ T cells was linked to a compartmentalized dissociation between effector programming and the transcription factor T-bet. In line with this, activation of LN CD8+ T cells only partially induced the acquisition of cytolytic functions relative to peripheral blood CD8+ T cells. These results suggest that a state of immune privilege against CD8+ T cell-mediated cytolysis exists in lymphoid tissue, potentially facilitating the persistence of HIV.

Investigating the Interplay between Sister Chromatid Cohesion and Homolog Pairing in Drosophila Nuclei

Following DNA replication, sister chromatids must stay connected for the remainder of the cell cycle in order to ensure accurate segregation in the subsequent cell division. This important function involves an evolutionarily conserved protein complex known as cohesin; any loss of cohesin causes premature sister chromatid separation in mitosis. Here, we examined the role of cohesin in sister chromatid cohesion prior to mitosis, using fluorescence in situ hybridization (FISH) to assay the alignment of sister chromatids in interphase Drosophila cells. Surprisingly, we found that sister chromatid cohesion can be maintained in G2 with little to no cohesin. This capacity to maintain cohesion is widespread in Drosophila, unlike in other systems where a reduced dependence on cohesin for sister chromatid segregation has been observed only at specific chromosomal regions, such as the rDNA locus in budding yeast. Additionally, we show that condensin II antagonizes the alignment of sister chromatids in interphase, supporting a model wherein cohesin and condensin II oppose each other’s functions in the alignment of sister chromatids. Finally, because the maternal and paternal homologs are paired in the somatic cells of Drosophila, and because condensin II has been shown to antagonize this pairing, we consider the possibility that condensin II-regulated mechanisms for aligning homologous chromosomes may also contribute to sister chromatid cohesion.

Identification and characterization of HIV-specific resident memory CD8+ T cells in human lymphoid tissue

Resident memory T cells in lymphoid tissues help restrain HIV replication in elite controllers. Taking residence to defend In HIV+ individuals receiving antiretroviral therapy, lymphoid tissues (LTs) that CD4+ T cells home to are a key site of HIV persistence. Studying the immune response to HIV in LTs has remained a challenge. By obtaining LTs from HIV+ individuals, Buggert et al. have carried out comprehensive transcriptional and epigenetic analyses on CD8+ T cells in these LTs. They report that CD8+ T cells in LTs of HIV+ individuals have a signature associated with resident memory T cells (TRMs) and that the frequency of these HIV-responsive LT-resident CD8+ T cells was considerably increased in elite controllers. The study brings to the fore the importance of HIV-specific LT-resident TRMs in restraining HIV replication in LTs. Current paradigms of CD8+ T cell–mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8+ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (TRMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8+ T cells in LTs also resemble TRMs. Moreover, high frequencies of HIV-specific CD8+ TRMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific TRMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-TRMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses resident within LTs.

OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes

Significance FISH enables researchers to visualize the subcellular distribution of RNA and DNA molecules in individual cells. The recent development of FISH methods employing probes composed of synthetic DNA oligonucleotides (oligos) allows researchers to tightly control aspects of probe design such as binding energy and genomic specificity. Although oligo FISH probes are central to many recently developed massively multiplexed and superresolution imaging methods, no dedicated computational utility exists to facilitate the design of such probes on the genome-wide scale. Here, we introduce a streamlined pipeline for the rapid, genome-scale design of oligo FISH probes and validate our approach by using conventional and superresolution imaging. Our method provides a framework with which to design oligo-based hybridization experiments. Oligonucleotide (oligo)-based FISH has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate new and existing utilities into the pipeline as desired, and introduces a method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised machine learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with diffraction-limited and single-molecule superresolution imaging of chromosomal and RNA targets. We anticipate that this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.

Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in Drosophila nuclei

Metazoan chromosomes are folded into discrete sub-nuclear domains, referred to as chromosome territories (CTs). The molecular mechanisms that underlie the formation and maintenance of CTs during the cell cycle remain largely unknown. Here, we have developed high-resolution chromosome paints to investigate CT organization in Drosophila cycling cells. We show that large-scale chromosome folding patterns and levels of chromosome intermixing are remarkably stable across various cell types. Our data also suggest that the nucleus scales to accommodate fluctuations in chromosome size throughout the cell cycle, which limits the degree of intermixing between neighboring CTs. Finally, we show that the cohesin and condensin complexes are required for different scales of chromosome folding, with condensin II being especially important for the size, shape, and level of intermixing between CTs in interphase. These findings suggest that large-scale chromosome folding driven by condensin II influences the extent to which chromosomes interact, which may have direct consequences for cell-type specific genome stability.

Human Immunodeficiency Virus Type-1 Elite Controllers Maintain Low Co-Expression of Inhibitory Receptors on CD4+ T Cells

Human immunodeficiency virus type-1 (HIV-1) elite controllers (ELCs) represent a unique population that control viral replication in the absence of antiretroviral therapy (cART). It is well established that expression of multiple inhibitory receptors on CD8+ T cells is associated with HIV-1 disease progression. However, whether reduced co-expression of inhibitory receptors on CD4+ T cells is linked to natural viral control and slow HIV-1 disease progression remains undefined. Here, we report on the expression pattern of numerous measurable inhibitory receptors, associated with T cell exhaustion (programmed cell death-1, CTLA-4, and TIGIT), on different CD4+ T cell memory populations in ELCs and HIV-infected subjects with or without long-term cART. We found that the co-expression pattern of inhibitory receptors was significantly reduced in ELCs compared with HIV-1 cART-treated and viremic subjects, and similar to healthy controls. Markers associated with T cell exhaustion varied among different memory CD4+ T cell subsets and highest levels were found mainly on transitional memory T cells. CD4+ T cells co-expressing all inhibitory markers were positively correlated to T cell activation (CD38+ HLA-DR+) as well as the transcription factors Helios and FoxP3. Finally, clinical parameters such as CD4 count, HIV-1 viral load, and the CD4/CD8 ratio all showed significant associations with CD4+ T cell exhaustion. We demonstrate that ELCs are able to maintain lower levels of CD4+ T cell exhaustion despite years of ongoing viral replication compared with successfully cART-treated subjects. Our findings suggest that ELCs harbor a “healthy” state of inhibitory receptor expression on CD4+ T cells that might play part in maintenance of their control status.

Highly Structured Homolog Pairing Reflects Functional Organization of the Drosophila Genome

Trans-homolog interactions encompass potent regulatory functions, which have been studied extensively in Drosophila, where homologs are paired in somatic cells and pairing-dependent gene regulation, or transvection, is well-documented. Nevertheless, the structure of pairing and whether its functional impact is genome-wide have eluded analysis. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, discovering that homologs pair relatively precisely genome-wide in addition to establishing trans-homolog domains and compartments. We also elucidated the structure of pairing with unprecedented detail, documenting significant variation across the genome. In particular, we characterized two forms: tight pairing, consisting of contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional role genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing. One Sentence Summary Haplotype-resolved Hi-C reveals structures of homolog pairing and global implications for gene activity in hybrid PnM cells.

Diversification and collapse of a telomere elongation mechanism

In virtually all eukaryotes, telomerase counteracts chromosome erosion by adding repetitive sequence to terminal ends. Drosophila melanogaster instead relies on specialized retrotransposons that insert preferentially at telomeres. This exchange of goods between host and mobile element—wherein the mobile element provides an essential genome service and the host provides a hospitable niche for mobile element propagation—has been called a ‘genomic symbiosis’. However, these telomere-specialized, ‘jockey’ family elements may actually evolve to selfishly over-replicate in the genomes that they ostensibly serve. Under this intra-genomic conflict model, we expect rapid diversification of telomere-specialized retrotransposon lineages and possibly, the breakdown of this tenuous relationship. Here we report data consistent with both predictions. Searching the raw reads of the 15-million-year-old ‘melanogaster species group’, we generated de novo jockey retrotransposon consensus sequences and used phylogenetic tree-building to delineate four distinct telomere-associated lineages. Recurrent gains, losses, and replacements account for this striking retrotransposon lineage diversity. Moreover, an ancestrally telomere-specialized element has ‘escaped,’ residing now throughout the genome of D. rhopaloa. In D. biarmipes, telomere-specialized elements have disappeared completely. De novo assembly of long-reads and cytogenetics confirmed this species-specific collapse of retrotransposon-dependent telomere elongation. Instead, telomere-restricted satellite DNA and DNA transposon fragments occupy its terminal ends. We infer that D. biarmipes relies instead on a recombination-based mechanism conserved from yeast to flies to humans. Combined with previous reports of adaptive evolution at host proteins that regulate telomere length, telomere-associated retrotransposon diversification and disappearance offer compelling evidence that intra-genomic conflict shapes Drosophila telomere evolution.

The genome-wide, multi-layered architecture of chromosome pairing in early Drosophila embryos

Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. Here, we focused on trans interactions in Drosophila, where homologous chromosomes are paired in somatic cells from embryogenesis through adulthood. We first addressed the long-standing question of whether pairing extends genome-wide and, to this end, developed a haplotype-resolved Hi-C approach that uses a new strategy to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This approach revealed striking genome-wide pairing in Drosophila embryos. Moreover, we discovered pairing to be surprisingly structured, with trans-homolog domains and interaction peaks, many coinciding with the positions of analogous cis features. We also found a significant correlation between pairing and the chromatin accessibility mediated by the pioneer factor Zelda. Our findings reveal a complex, highly structured organization underlying homolog pairing, first discovered more than a century ago. One Sentence Summary A robust approach for haplotype-resolved Hi-C reveals highly-structured homolog pairing in early stage Drosophila embryos.