Epigenetic mechanisms, T-cell activation, and CCR5 genetics interact to regulate T-cell expression of CCR5, the major HIV-1 coreceptor.

CAPRISA, 2015.Abstract available in pdf

Silent IL2RG Gene Editing in Human Pluripotent Stem Cells.

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes

Comparative genomics of primate CCL3L and CCL4L loci.

<p>(A) Comparison of <i>CCL3L</i> and <i>CCL4L</i> in human and nonhuman primates. The top panel shows a schema of the chemokine locus at human chromosome 17q12 based on the NT_010799.14 contig. <i>CCL3</i> and <i>CCL4</i> exist as single-copy genes per haploid genome. The genes encoding the non-allelic isoforms of <i>CCL3</i> (National Center for Biotechnology Information gene ID given in parentheses) are denoted as <i>CCL3L1</i> (6349), <i>CCL3L2</i> (390788), and <i>CCL3L3</i> (414062) and those of <i>CCL4</i> are denoted as <i>CCL4L1</i> (9560) and <i>CCL4L2</i> (388372). The middle panel shows a schema of the <i>CCL3L</i> and <i>CCL4L</i> locus in chimpanzee based on the chromosome 17NW_001226927.1 contig. <i>CCL3L</i> orthologs (denoted as “1” and “2”) map ∼ 1.6 Mb apart in this contig. In contrast to the human locus, chimpanzee contigs lack <i>CCL3L2</i>. The bottom panel shows a schema of the <i>CCL3L</i> and <i>CCL4L</i> locus in rhesus monkey based on chromosome 16 NW_001103987 contig. Of note, other orthologs of <i>CCL3L</i> and <i>CCL4L</i> were found in two other rhesus contigs (NW_001103644.1 and NW_001102959). CpG islands found in primate <i>CCL3L</i> and <i>CCL4L</i> loci are also depicted. Distances between genes are approximate, and the map is not to scale. The arrows denote the orientation of the genes. k, kb; M, Mb. (B and C) Schematic representation of genomic and mRNA structure of human <i>CCL3L</i> and <i>CCL4L</i> genes that have mRNA splicing patterns that are similar (B) or dissimilar (C) to <i>CCL3</i> and <i>CCL4.</i> Exons are represented as boxes and introns as connecting lines labeled with Roman numbers; the splicing pattern is denoted by the dashed lines. <i>CCL3L1</i>, <i>CCL3L3</i>, and <i>CCL4L2</i> are each composed of three exons, and the start codon (denoted with an arrow) is located in the first exon. <i>CCL4L1</i> has a transition in the splicing acceptor site located in intron II (AG→GG, indicated in red), which results in the generation of aberrantly spliced transcripts that use alternative acceptor sites located either in the intron II or in the third exon <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-Colobran1" target="_blank">[26]</a>. <i>CCL3L2</i> was previously considered as a pseudogene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-Menten1" target="_blank">[5]</a>. However, recent studies in our lab suggest that it has a four exon structure and is predicted to transcribe alternatively spliced mRNA species with open reading frames (ORFs) that contain chemokine-like domains <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-ShostakovichKoretskaya1" target="_blank">[16]</a>; CCL3L2 mRNA transcripts originate from two novel upstream exons (designated as 1A and 1B) and are linked to the second and third exons, which are homologous to exons 2 and 3 found in <i>CCL3L1</i> or <i>CCL3L3</i>. (D) Nucleotide sequence of human <i>CCL3L1</i> (or <i>CCL3L3</i>) and its alignment with four distinct chimpanzee <i>CCL3L</i> (<i>chCCL3L</i>) orthologous genes from the translation initiation site until the start of intron 1. The translational start codon in <i>hCCL3L1</i> is underlined. Horizontal arrows delimit the exon–intron boundaries. Dashes indicate deletions. Polymorphic sites relative to the <i>hCCL3L1</i> are shown in red. The vertical arrow represents the site for signal peptidase cleavage. <i>chCCL3L ortholog 1</i> is predicted to encode a chemokine with amino acids that are shared with both hCCL3L1 and hCCL3. <i>chCCL3L ortholog 2</i> has a deletion of 17 nucleotides (relative to <i>hCCL3L1</i>) that may lead to loss of the signal peptide cleavage motif. Notably, two additional and different <i>CCL3L</i> orthologs were found in two independent chimpanzee contigs, denoted as NW_001227489.1 (ortholog 3) and NW_001227474.1 (ortholog 4), which have a mutation at the translation initiation site (shown in purple) and differ from each other in the splicing donor site of intron 1 (shown in blue) and other genomic regions (unpublished data). Of note, all four <i>chCCL3L1</i> orthologs had sequences that were completely homologous to the primer–probe sets used to detect <i>CCL3L</i> CNV in humans and chimpanzee previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-Gonzalez1" target="_blank">[12]</a> and by Degenhardt et al. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000359#pgen.1000359-Degenhardt1" target="_blank">[9]</a>. All the chimpanzee orthologs are also predicted to encode transcripts with potential ORFs with chemokine-like domains. The accession numbers for the predicted ORFs encoded by chimpanzee <i>CCL3L</i> orthologs 1, 2, 3, and 4 are NP_001029254, XP_001152451, XP_001172388, and XP_001172226, respectively.</p

A cohort-based study of host gene expression: tumor suppressor and innate immune/inflammatory pathways associated with the HIV reservoir size

&lt;p&gt;The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). Most prior studies of host genetic predictors of HIV control have focused on "elite controllers," rare individuals able to control virus in the absence of ART. However, there have been few genetic studies among ART-suppressed non-controllers, who make up the majority of people living with HIV (PLWH). We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 HIV+ ART-suppressed non-controllers. After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, &lt;em&gt;P3H3&lt;/em&gt; and &lt;em&gt;NBL1&lt;/em&gt;, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (&lt;em&gt;KCNJ2&lt;/em&gt;, &lt;em&gt;GJB2&lt;/em&gt;), inflammasome (&lt;em&gt;IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10&lt;/em&gt;), and innate immunity (TLR7) genes (FDR-adjusted q&lt;0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q=0.006), IL-1/NRLP3 inflammasome (q=0.008), and IL-10 (q=0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-a protein associations achieving statistical significance (p&lt;0.05). Of note, plasma IL-10 was also significantly inversely associated with HIV DNA (p=0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. Further data are needed to validate these findings, including functional genomic studies, larger cohorts including underrepresented PLWH in research, and those including dedicated assays to measure the replication-competent HIV reservoir.&lt;/p&gt;&lt;p&gt;Funding provided by: National Institute of Allergy and Infectious Diseases&lt;br&gt;Crossref Funder Registry ID: https://ror.org/043z4tv69&lt;br&gt;Award Number: K23GM112526&lt;/p&gt;&lt;p&gt;Funding provided by: National Institute of Allergy and Infectious Diseases&lt;br&gt;Crossref Funder Registry ID: https://ror.org/043z4tv69&lt;br&gt;Award Number: U19 AI096109&lt;/p&gt;&lt;p&gt;Funding provided by: National Institute of Allergy and Infectious Diseases&lt;br&gt;Crossref Funder Registry ID: https://ror.org/043z4tv69&lt;br&gt;Award Number: UM1 AI126623&lt;/p&gt;&lt;p&gt;Funding provided by: National Center for Advancing Translational Sciences&lt;br&gt;Crossref Funder Registry ID: https://ror.org/04pw6fb54&lt;br&gt;Award Number: KL2TR002317&lt;/p&gt;&lt;p&gt;Funding provided by: American Foundation for AIDS Research&lt;br&gt;Crossref Funder Registry ID: https://ror.org/02jesmk44&lt;br&gt;Award Number: 108072-50-RGRL&lt;/p&gt;&lt;p&gt;Funding provided by: Bill &amp; Melinda Gates Foundation&lt;br&gt;Crossref Funder Registry ID: https://ror.org/0456r8d26&lt;br&gt;Award Number: INV-008500&lt;/p&gt;&lt;p&gt;Funding provided by: National Institute of Allergy and Infectious Diseases&lt;br&gt;Crossref Funder Registry ID: https://ror.org/043z4tv69&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;HIV+ ART-suppressed non-controllers from the UCSF SCOPE and Options HIV+ cohorts were included in the study.&lt;/p&gt; &lt;p&gt;Cryopreserved PBMCs were enriched for CD4+ T cells (StemCell, Vancouver, Canada), and RNA was extracted from CD4+ T cells using the AllPrep Universal Kit (Qiagen, Hilden, Germany) with one aliquot set aside for HIV reservoir quantification and a second aliquot for host RNA sequencing. Host RNA sequencing was analyzed using the HTStream pre-processing pipeline (s4hts.github.io/htstream/) was used for removing PCR duplicates, adapters, N characters, PolyA/T sequences, Phix contaminants, and poor-quality sequences (with quality score &lt;20 with sliding window of 10 base pairs). The quality of raw reads was assessed using FastQC. All samples had a per base quality score and sequence quality score &gt;30. RNA-seq reads were then mapped to the human genome (GRCh38) with a corresponding annotation file from the GENCODE project. Alignment and gene quantification were performed using the STAR alignment tool and its quantification protocol. Gene expression was converted to counts per million (CPM). The mean-variance trend was estimated to assign observational weights based on predicted variance on log&lt;sub&gt;2&lt;/sub&gt;-counts per million (log&lt;sub&gt;2&lt;/sub&gt;-CPM) using the Limma-Voom pipeline.&lt;/p&gt; &lt;p&gt;For validation of intracellularly expressed or membrane-associated encoded proteins, we performed ELISA from peripheral CD4-enriched T cells. CD4+ T cells were isolated from PBMC by negative selection, using the EasySep Human CD4+ T Cell Isolation Kit (StemCell Technologies, Vancouver, BC, Canada), following manufacturer's guidelines. The Muse Human CD4 T Cell kit (Luminex, Austin, TX) in combination with the Guava Muse Cell Analyzer was used to determine the concentration and percentages of CD4+ T cells after the isolation. To generate cellular lysates, purified CD4+ T cells were subjected to three cycles of freezing/thawing, using a dry ice (frozen CO&lt;sub&gt;2&lt;/sub&gt;) /absolute ethanol mixture and a 37˚C water bath. Complete lysis was verified by trypan blue staining and microscopic analysis. Lysates were spun down at 1,500 g for 10 min at 4˚C (to remove cellular debris) and the supernatants diluted 1:5 with PBS and kept at -80˚C until the time of protein quantification. Total protein concentration of the CD4+ T cell lysates was determined using the Pierce®BCA assay (Thermo Fisher Scientific). The mean value obtained was 0.94 mg/ml (range: 0.66 mg/ml – 1.18 mg/ml). Chemiluminescence or absorbance was read on a SpectraMax® iD5 multi-mode plate reader (Molecular Devices, San Jose, CA) and reported in relative light units (RLU). A standard curve was constructed by plotting the log mean RLU reading for each standard on the y-axis against the log of known concentrations on the x-axis using the SoftMax Pro 7.1 software (Molecular Devices, San Jose, CA). Data were normalized by total protein concentration to accurately reflect the total population of cells (live and dead). Briefly, a 1:5 dilution factor (based on supernatant dilution with PBS at the time of CD4+ T cell isolation) was used to calculate the concentration in the lysate before the dilution. Each protein marker was then quantified using the marker-specific ELISA (again, taking into account the 1:5 dilution performed before cryopreservation of the lysates). Data normalization was performed by dividing the concentration of each protein in the final lysate (e.g., for P3H3 in pg/ml) by the total protein concentration (mg/ml).&lt;/p&gt; &lt;p&gt;For validation of the several inflammatory pathway genes identified in association with HIV usRNA, most of the encoded proteins were secreted proteins, and thus, we performed high-sensitivity multiplex plasma cytokine quantification (Meso Scale Diagnostics). Plasma levels of IP-10 (the encoded protein for &lt;em&gt;CXCL10&lt;/em&gt;), G-CSF (&lt;em&gt;GCSF&lt;/em&gt;) and pentraxin 3 (&lt;em&gt;TNFAIP5&lt;/em&gt;) were quantified using the electrochemiluminescence-based 3-plex mesoscale discovery (MSD) platform (U-Plex mesoscale discovery, Rockville, MA); IL-10 (&lt;em&gt;IL10&lt;/em&gt;), IL-1β (&lt;em&gt;IL1B&lt;/em&gt;) and TNF-α (&lt;em&gt;TNFA&lt;/em&gt;) were measured in a separate 3-plex S-plex Proinflammatory panel kit, and IL‑1α (&lt;em&gt;IL1A&lt;/em&gt;) was quantified by a V-plex kit. In all these assays, undiluted samples were run in duplicate following manufacturer's instructions, and protein concentrations were determined using MSD Discovery Workbench (version 4.0.13) analysis software. The light intensities from the samples were interpolated using a four-parameter logistic fit (FourPL) to a standard curve of electrochemiluminescence generated from eight calibrators of know concentrations. The lower limit of detection for each marker can be found on the manufacturer's website (MesoScale Diagnostics, &lt;a href="https://www.mesoscale.com/~/media/files/handout/assaylist.pdf"&gt;https://www.mesoscale.com/~/media/files/handout/assaylist.pdf&lt;/a&gt;).&lt;/p&gt

Silent IL2RG Gene Editing in Human Pluripotent Stem Cells

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes