Identification of Chromatin Regulators Perturbed in Hematopoietic Stem and Progenitor Cell Aging

As lifespan is increasing globally, there is a critical need to identify strategies to extend healthspan and prevent chronic diseases into older age. The long-term goal of my research is to identify novel strategies to ameliorate aging-induced decline in hematopoietic stem cell (HSC) function. HSCs give rise to all mature blood and immune cells. With age, HSCs undergo defects in their differentiation ability which correlates with a decline in immune function. Comprehensive knowledge of gene regulatory and epigenetic mechanisms underlying this defect is a barrier to developing therapies to ameliorate aging-associated decline in HSC function. Therefore, my project focuses on understanding the gene regulatory mechanisms underlying this decline in HSC function. Before delving into the gene regulatory mechanisms that go awry with age, it is important to identify which mechanisms are important for the differentiation of HSCs to mature cells. The majority of screening approaches for the identification of novel genes and gene regulatory elements rely on robust in vitro assays. In my thesis work, I have demonstrated in my thesis work that one such assay widely used in the field to differentiate hematopoietic stem and progenitor cells (HSPCs) to B-lymphoid cells performs in a qualitative rather than a quantitative manner which provides implications for interpretations of results this assay. Also, by mining publicly available gene expression data sets and data from an unpublished shRNA knockdown screen, I have identified that the epigenetic regulator lysine acetyltransferase 6b (Kat6b) is important for HSC function as well as demonstrated that KAT6B levels are significantly decreased in expression in aged long term-hematopoietic stem cells (LT-HSCs) at the transcript and protein levels, using qPCR and immunofluorescence. In addition, I have observed that knockdown of Kat6b leads to enhanced myeloid differentiation from LT-HSCs by using in vitro and in vivo assays which partially replicates aging-associated hematopoietic phenotypes. Transcriptome analysis suggests that Kat6b knockdown in LT-HSCs leads to dysregulation of differentiation signatures and an increase in inflammation. These data support increasing the levels of Kat6b as a novel therapeutic strategy for ameliorating aging-associated hematopoietic decline

Precision Cas9 Genome Editing in vivo with All-in-one, Self-targeting AAV Vectors [preprint]

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes

Aging-associated decrease in the histone acetyltransferase KAT6B is linked to altered hematopoietic stem cell differentiation.

Aged hematopoietic stem cells (HSCs) undergo biased lineage priming and differentiation toward production of myeloid cells. A comprehensive understanding of gene regulatory mechanisms causing HSC aging is needed to devise new strategies to sustainably improve immune function in aged individuals. Here, a focused short hairpin RNA screen of epigenetic factors reveals that the histone acetyltransferase Kat6b regulates myeloid cell production from hematopoietic progenitor cells. Within the stem and progenitor cell compartment, Kat6b is highly expressed in long-term (LT)-HSCs and is significantly decreased with aging at the transcript and protein levels. Knockdown of Kat6b in young LT-HSCs causes skewed production of myeloid cells at the expense of erythroid cells both in vitro and in vivo. Transcriptome analysis identifies enrichment of aging and macrophage-associated gene signatures alongside reduced expression of self-renewal and multilineage priming signatures. Together, our work identifies KAT6B as a novel epigenetic regulator of hematopoietic differentiation and a target to improve aged immune function

A cohesin traffic pattern genetically linked to gene regulation [preprint]

Cohesin-mediated loop extrusion folds interphase chromosomes at the ten to hundreds kilobases scale. This process produces structural features such as loops and topologically associating domains. We identify three types of cis-elements that define the chromatin folding landscape generated by loop extrusion. First, CTCF sites form boundaries by stalling extruding cohesin, as shown before. Second, transcription termination sites form boundaries by acting as cohesin unloading sites. RNA polymerase II contributes to boundary formation at transcription termination sites. Third, transcription start sites form boundaries that are mostly independent of cohesin, but are sites where cohesin can pause. Together with cohesin loading at enhancers, and possibly other cis-elements, these loci create a dynamic pattern of cohesin traffic along the genome that guides enhancer-promoter interactions. Disturbing this traffic pattern, by removing CTCF barriers, renders cells sensitive to knock-out of genes involved in transcription initiation, such as the SAGA and TFIID complexes, and RNA processing such DEAD-Box RNA helicases. In the absence of CTCF, several of these factors fail to be efficiently recruited to active promoters. We propose that the complex pattern of cohesin movement along chromatin contributes to appropriate promoter-enhancer interactions and localization of transcription and RNA processing factors to active genes

Synthetic sterility and RNAi deficiency in <i>elli-1</i>.

<p>A) Larval arrest on control (+) and <i>his-44</i> (-) RNAi showing <i>elli-1(sam3)</i> enhances the RNAi resistant phenotype of <i>drh-3(sam27)</i> at permissive temperature (20°C). Same colored box plots represent duplicate experiments performed on different weeks. B) Brood sizes show that <i>elli-1(sam3)</i> enhances sterility (no brood) of <i>drh-3(sam27)</i> at permissive temperature. C) STYO14 staining of dissected germlines shows RNA pooling around the nuclear periphery and in the rachis of both <i>csr-1</i> and <i>elli-1</i> mutants. D) Model for CSR-1 and ELLI-1 function. P granules (yellow) reside on the nuclear periphery where they receive transcripts coming through the nuclear pore complex. The CSR-1 complex functions in P granules to recognize germline abundant or licensed transcripts. Our model is that CSR-1 and ELLI-1 function to move germline abundant or licensed transcripts through and away from P granules and into the cytoplasm. In mutants this dispersal of RNA is blocked, RNA and P granules accumulate, and fertility and RNAi efficacy is decreased. It remains unclear whether ELLI-1 directly interacts with RNA.</p

<i>elli-1</i> expression.

<p>A) Fluorescence In Situ Hybridization (FISH) of <i>elli-1</i> mRNA (red) shows germline expression. DAPI/DNA is blue. B) <i>elli-1</i>::<i>GFP</i>::<i>3xFLAG</i> showing diffuse cytoplasmic expression with some ELLI-1 foci in a dissected germline. 4.3% of ELLI-1 foci are docked to P granules (arrowheads), but most of these foci are in the central shared cytoplasm of the rachis. C) ELLI-1::GFP partially overlaps with the expression of the P-body component CGH-1 (red), primarily in the rachis instead of at the nuclear periphery of germ cells. D) <i>elli-1</i>::<i>GFP</i>::<i>3xFLAG</i> expression in primordial germ cells (arrows) during embryogenesis and the first larval stage. E) Embryonic lethality associated in <i>elli-1</i> worms.</p

Gene expression analysis of <i>elli-1</i>.

<p>A) Volcano plot showing the fold change and significance of gene expression in <i>elli-1</i>. Significantly regulated genes above or below 1.2-fold are shown in red, with a subset of soma-enriched genes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006611#pgen.1006611.ref043" target="_blank">43</a>] in green (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006611#pgen.1006611.s001" target="_blank">S1 Table</a>). B) Proportional Venn diagrams comparing overlap between the 1079 <i>elli-1</i> regulated genes and previously published <i>csr-1</i> and <i>pgl-1</i> regulated [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006611#pgen.1006611.ref013" target="_blank">13</a>], CSR-1 target [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006611#pgen.1006611.ref004" target="_blank">4</a>], and soma and germline enriched [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006611#pgen.1006611.ref043" target="_blank">43</a>] datasets. C) Increased average expression of CSR-1 complex and core P-granule components in the <i>elli-1</i> expression array (black) and <i>elli-1</i> qRTPCR (green). Pink line indicates the arbitrary 1.2-fold increased expression cutoff. Increased <i>ego-1</i> and <i>ekl-1</i> expression was statistically significant, but under the 1.2-fold cutoff. D) <i>elli-1</i> Volcano plot showing increased expression of genes required for RNAi-dependent gene silencing (green).</p

Enlarged P-granule alleles from screen.

<p>Enlarged P-granule alleles from screen.</p

<i>elli-1</i> alleles and phenotypes.

<p>A) Location and flanking sequences of <i>elli-1</i> alleles. Red lines show early stop codons, grey lines show base pair substitutions and deletions. Yellow boxes indicate a codon in the reading frame. ELLI-1’s N-terminal predicted disordered region is shown by the green bars. B) <i>elli-1(sam3)</i> and <i>elli-1(RNAi)</i> cause bright expression of enlarged PGL-1::GFP granules. C) Fosmid rescue of bright PGL-1::GFP expression in <i>elli-1(sam3)</i> animals. Red body-wall muscles mark animals with the rescuing fosmid. D) Cross-section through pachytene germ cells on the surface of wild-type and <i>elli-1(sam3)</i> germlines stained with anti-PGL-1 (green), anti-Nuclear Pore Complex antibody mAb414 (red), and DAPI/DNA (blue). P granules in <i>csr-1</i> and <i>elli-1</i> germlines are enlarged and often detach from the nuclear periphery. E) Cross-section through <i>wild-type</i> and <i>elli-1</i> germlines with the same strains as in D. Arrows show PGL-1 accumulation in the shared cytoplasm. F) GFP tagging endogenous GLH-1 in germlines of <i>wild-type</i> and <i>elli-1(sam3)</i> worms.</p

Screen for regulators of P-granule accumulation.

<p>A) Screening strategy to isolate mutants with enlarged PGL-1::GFP granules. PGL-1::GFP worms were mutagenized, 2000 F1 progeny were cloned to individual plates, and F2 progeny were screened for homozygous (m/m) mutants with enlarged P granules B) PGL-1::GFP expression in the gonad arm of parental (P0) and mutant strains. C) Relative PGL-1 intensity in mutant germlines normalized to the parental control. D) Increased PGL-1::GFP size in mutants correlates with increased PGL-1 intensity.</p