ALBERT

Description: Tactile-foraging ducks are specialist birds known for their touch-dependent feeding behavior. They use dabbling, straining, and filtering to find edible matter in murky water, relying on the sense of touch in their bill. Here, we present the molecular characterization of embryonic duck bill, which we show contains a high density of mechanosensory corpuscles innervated by functional rapidly adapting trigeminal afferents. In contrast to chicken, a visually foraging bird, the majority of duck trigeminal neurons are mechanoreceptors that express the Piezo2 ion channel and produce slowly inactivating mechano-current before hatching. Furthermore, duck neurons have a significantly reduced mechano-activation threshold and elevated mechano-current amplitude. Cloning and electrophysiological characterization of duck Piezo2 in a heterologous expression system shows that duck Piezo2 is functionally similar to the mouse ortholog but with prolonged inactivation kinetics, particularly at positive potentials. Knockdown of Piezo2 in duck trigeminal neurons attenuates mechano current with intermediate and slow inactivation kinetics. This suggests that Piezo2 is capable of contributing to a larger range of mechano-activated currents in duck trigeminal ganglia than in mouse trigeminal ganglia. Our results provide insights into the molecular basis of mechanotransduction in a tactile-specialist vertebrate.

Description: Pomalidomide, a second-generation immunomodulatory drug, is a fetal hemoglobin (HbF) inducing agent with potential implications for the treatment of β-hemoglobinopathies such as sickle cell disease (SCD). However, its mechanism of action remains unknown. Through an in-depth characterization of human erythropoiesis and globin gene regulatory networks, we now provide evidence that pomalidomide alters transcription networks involved in erythropoiesis and globin switching, thereby leading to a partial reprogramming of adult hematopoietic progenitors toward fetal-like erythropoiesis. Adult peripheral blood CD34+ cells from normal individuals were differentiated toward the red cell lineage using an adapted 3-phase culture system. At day 14 of culture, we observed a reciprocal globin gene switch at the mRNA and protein levels. These results were confirmed by high performance liquid chromatography of hemolysates (HbF/(HbF+HbA): 31.7 ± 1.4% vs. 6.5 ± 0.7% pomalidomide and vehicle, respectively). Next, we studied erythroid differentiation using flow cytometric analyses of the cell surface markers interleukin-3R (IL-3R), glycophorin A (GPA), CD34 and CD36 for early erythroid precursors (BFU-E and CFU-E) as well as GPA, α4-integrin and band3 for terminal erythroid differentiation. While there were no changes in terminal erythroblast maturation, an accumulation of BFU-E in pomalidomide-treated cultures at days 2 and 4 of differentiation was seen, indicating a delay at the BFU-E to CFU-E transition, and also, that pomalidomide exerts its effect in the early-stages of erythropoiesis. Indeed, treatment with pomalidomide during the phase of the culture system that generates erythroid progenitors led to significantly more γ-globin expression than treatment during the phase which proerythroblasts undergo terminal erythroid differentiation. At the molecular level, pomalidomide was found to rapidly and robustly decrease Ikaros (IKZF1) expression exclusively by post-translational targeting to the proteasome. Moreover, pomalidomide selectively reduced the expression of components of key globin regulatory pathways including BCL11A, SOX6, KLF1, GATA1 and LSD1 while not affecting others (e.g. CoREST, GATA2, GFI1B, and HDAC1). Pomalidomide had a transient effect on GATA1 and KLF1 expression. While shRNA knockdown of Ikaros using two different lentiviral constructs delayed erythroid differentiation, it failed to appreciably stimulate HbF production or alter BCL11A expression. These results suggest that the loss of Ikaros alone is insufficient to recapitulate the phenotype observed in pomalidomide-treated conditions. We next compared the expression levels of proteins involved in globin gene regulation among untreated peripheral blood, pomalidomide-treated peripheral blood and untreated cord blood-derived erythroid cells. We found striking similarities between cord blood and pomalidomide-treated adult cells at day 4 of differentiation. Indeed, BCL11A, KLF1, SOX6, LSD1 and GATA1 showed decreased expression levels both in cord blood and pomalidomide-treated adult peripheral blood, while the levels of CoREST, HDAC1 and GATA2 remained unchanged indicating that pomalidomide partially reprograms adult erythroid cells to a fetal-like state. Taken together, our results show that the mechanism underlying reactivation of HbF by pomalidomide involves Ikaros-independent reprogramming of adult erythroid progenitors. Finally, we found that this mechanism is conserved in SCD-derived CD34+ cells. Our work has broad implications for globin switching, as we provide direct evidence that Ikaros does not play a major role in the repression of γ-globin during adult erythropoiesis, and further supports the previously held notion that globin chain production is determined prior to or at the level of CFU-E. Disclosures Allen: Celgene: Research Funding; Bristol Myers Squibb: Equity Ownership; Onconova: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Description: The maturation of a committed erythroid progenitor to a functional erythrocyte is characterized by a global decline in transcription and progressive nuclear condensation that ultimately culminates in enucleation. At the molecular level, erythroid maturation is driven by erythroid transcription factors and chromatin modifying enzymes that work in a coordinated manner to drive the expression of erythroid-specific genes, while silencing most other genes during the process of chromatin condensation and enucleation. Setd8 is the sole histone methyltransferase in mammals capable of mono-methylating histone H4 Lysine 20 (H4K20me1). In vitro studies suggest that Setd8 and H4K20me1 play critical roles in cell cycle progression, nuclear condensation, and DNA damage response (Reviewed in Beck et al, Genes and Development, 2012). In vivo studies on the function of Setd8 and H4K20me1 have been limited by the early embryonic lethality of constitutive Setd8 deletion (Oda et al, MCB, 2009). Although Setd8 is broadly expressed in tissues, there is a striking increase in Setd8 expression in CD71+ erythroid cells (Wu et al, Genome Biology, 2009), suggesting that Setd8 may have erythroid specific functions. Initial studies from our lab and others suggest that Setd8 regulates erythroid maturation and represses Gata2 expression (Malik et al MCB 2015; DeVilbiss et al MCB 2015). To delineate the function of Setd8 in vivo, we generated an erythroid-specific Setd8 deletion by crossing mice with flox sites flanking exon 7 of Setd8 (Setd8 fl/fl; Oda et al, MCB, 2009) with mice expressing a Cre-Recombinase GFP fusion protein under the control of the endogenous erythropoietin receptor promoter (ErGFPCre; Heinrich et al, Blood, 2004). Setd8Δ/Δ;ErGFPCre embryos demonstrated visible anemia starting at E9.5, with death occurring at E12.5 due to severe anemia. The early onset of anemia is consistent with a defect in primitive erythropoiesis. Cytospins and imaging flow cytometric analyses demonstrated a block in primitive erythroblast maturation and a profound impairment in nuclear condensation, with a nuclear area of 96 um2 in the Setd8 null erythroblasts and 56um2 in the littermate control erythroblasts at E10.5. Setd8 null erythroblasts also had abnormalities in cell cycle progression as well as increased staining for γH2AX, suggesting accumulation of DNA damage. There were similar numbers of Erythro-myeloid progenitors, (EMP; defined as defined as Ter119-negative, kit-hi,CD41-hi cells) in both the Setd8Δ/Δ;ErGFPCre and littermate control embryos, however the Setd8 null progenitors failed to differentiate into definitive erythroblasts. The role of Setd8 in transcriptional regulation is controversial, with some studies suggesting it is an activator and others suggesting it is a repressor. Global transcriptome analyses on sorted populations of Setd8 null and littermate control erythroblasts suggest that Setd8 acts primarily as a repressor in erythroid cells, with the majority of transcripts upregulated following Setd8 deletion. RNA-seq analyses further demonstrated a profound increase in the expression of cell cycle checkpoint enzymes such as P21 (fold change 73, p〈 e-53) and a significant increase in Gata2 expression (fold change 16, P

Description: Pediatric non-Down Syndrome acute megakaryoblastic leukemia (AMKL), a disease where megakaryocyte (MK) maturation is blocked, has a very poor prognosis. In 13% of AMKL cases of this type, the transcriptional co-factor MRTFA is expressed as part of a fusion protein. Normally, MRTFA levels increase in hematopoietic cells during megakaryopoiesis, making it likely that expression of MRTFA as part of a fusion protein leads to aberrant gene regulation, which results in leukemia. However, the mechanism by which megakaryopoiesis is blocked in AMKL is unknown. Therefore, we sought to parse out the role of MRTFA in normal megakaryopoiesis, so that we could better understand the cause for maturation block in leukemia. MRTFA is a co-factor of serum response factor (SRF). Knockout of either SRF or MRTFA in mice decreases MK maturation causing thrombocytopenia; and MRTFA overexpression (MRTFAOE) promotes MK maturation of primary human bone marrow (BM) cells in vitro. Our novel study shows that genomic regulation by MRTFA promotes MK maturation. In the human erythroleukemia (HEL) cell line, MRTFAOE enhances phorbol ester (TPA)-induced megakaryopoiesis, mimicking the effects of MRTFA on primary MK maturation. TPA-induced HEL cells with MRTFAOE achieve significantly higher 8N and 16N ploidy (p 〈 0.001, N = 4), compared to those without MRTFAOE. To identify the mechanisms underlying megakaryocytic differentiation, we bioinformatically analyzed anti-SRF chromatin immunoprecipitation (ChIP)-sequencing [approx. 15 million reads/sample] and RNA-sequencing data [approx. 20 million reads/sample], from non-induced and TPA-induced HEL cells, with and without MRTFAOE (N = 2/condition). We identified SRF peaks that change during TPA-induction, with and without MRTFAOE, and analyzed the strength of their binding and motif status. MRTFOE not only increased SRF binding to the genome during MK maturation (p ≤ 10-8), but preferentially retained binding at genomic CArG (CC[W]6GG) motifs, where SRF binds to in association with either MRTFA or specific ETS proteins (ELK1, ELK4). We then analyzed upregulated and downregulated genes during TPA-induction, with and without MRTFAOE, and identified those that had associated SRF peaks. As expected, TPA-induction upregulated megakaryocytic and cytoskeletal genes (VWF, ACTN1, CORO1A) and downregulated erythroid genes (KLF1, GYPB, GYPE). Interestingly, TPA-induction with MRTFAOE increased the number of upregulated genes by 27% and the number of downregulated genes by 10%. In TPA-induced cells, MRTFAOE increased the percentage of differentially expressed genes that had SRF peaks (25% versus 11% for upregulated genes, and 9% versus 4% for downregulated genes), further highlighting the direct role that MRTFA plays in MK maturation. Also, genes upregulated by TPA-induction alone have both ETS and CArG motifs, whereas those upregulated by TPA-induction with MRTFAOE lack ETS binding motifs, suggesting that MRTFAOE skews SRF binding toward CArG motifs. With anti-MRTFA ChIP-PCR in HEL cells, we confirmed the novel finding that along with SRF, MRTFA binds to regions associated with megakaryocytic and cytoskeletal genes (XRK6, CORO1A). Therefore, SRF and MRTFA together regulate expression of genes that are important for normal megakaryopoiesis, which explains why lack of these proteins adversely affects megakaryopoiesis in mice. We asked whether our findings in HEL cells were applicable to the more clinically relevant primary human BM cells. Anti-SRF and anti-MRTFA ChIP-PCR on day 0 primary human cells (CD34+ cells) and day 8 differentiated cell subpopulations (CD41+CD42-, CD41+CD42+) confirmed that both SRF and MRTFA have increased binding during megakaryopoiesis at target sites associated with upregulated genes, such as CORO1A, TNS1, and XRK6 (p 〈 0.001). Therefore, we illustrated that transcriptional regulation by SRF/MRTFA function similarly in human BM cells undergoing megakaryopoiesis. We show for the first time that MRTFA increases both the genomic association and activity of SRF, and upregulates genes that enhance primary human megakaryopoiesis. These findings suggest that aberrant expression of MRTFA as a fusion protein in AMKL may disrupt essential transcriptional regulation via the SRF/MRTFA axis, resulting in blocked MK maturation. This forms a crucial reference point for future studies to understand the altered SRF/MRTFA function in AMKL. Disclosures No relevant conflicts of interest to declare.

Description: Key Points PK deficiency manifests a broad spectrum in anemia severity that moderately improves after splenectomy. Close attention to monitoring for iron overload, gallstones, and other complications is recommended in all patients with PK deficiency.

Description: The erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, was the first hematopoietic niche discovered. The identity of EBI macrophages has thus far remained elusive. Given that Epo is essential for erythropoiesis and that Epor is expressed in numerous nonerythroid cells, we hypothesized that EBI macrophages express Epor so that Epo can act on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis. To test this notion, we used Epor-eGFPcre knockin mouse model. We show that in bone marrow (BM) and fetal liver, a subset of macrophages express Epor-eGFP. Imaging flow cytometry analyses revealed that 〉90% of native EBIs comprised F4/80+Epor-eGFP+ macrophages. Human fetal liver EBIs also comprised EPOR+ macrophages. Gene expression profiles of BM F4/80+Epor-eGFP+ macrophages suggest a specialized function in supporting erythropoiesis. Molecules known to be important for EBI macrophage function such as Vcam1, CD169, Mertk, and Dnase2α were highly expressed in F4/80+Epor-eGFP+ macrophages compared with F4/80+Epor-eGFP− macrophages. Key molecules involved in iron recycling were also highly expressed in BM F4/80+Epor-eGFP+ macrophages, suggesting that EBI macrophages may provide an iron source for erythropoiesis within this niche. Thus, we have characterized EBI macrophages in mouse and man. Our findings provide important resources for future studies of EBI macrophage function during normal as well as disordered erythropoiesis in hematologic diseases such as thalassemia, polycythemia vera, and myelodysplastic syndromes.

Description: Key Points TET3 knockdown impairs terminal erythroid differentiation, whereas TET2 knockdown leads to accumulation of erythroid progenitors. Global levels of 5mC are not altered by knockdown of either TET2 or TET3.

Description: Maturation of erythroid progenitors is associated with significant changes in gene expression in the context of a nucleus that dramatically decreases in size in preparation for enucleation, and is regulated by the coordinated action of transcriptional regulators and epigenetic modifiers. In eukaryotes, all DNA is bound by histone proteins into chromatin. Posttranslational modifications of the N-terminal "tails" of these proteins are key regulators of chromatin structure and gene expression. We hypothesized that terminal erythroid maturation is associated with changes in the abundance of specific histone posttranslational modifications. To address this hypothesis, we utilized mass spectrometry to perform an unbiased assessment of the abundance histone post translational modifications in maturing erythroblasts. We cultured peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) down the erythroid lineage using a semi-synchronous culture system (as outlined in Gautier et al. Cell Reports 2016), and sent cells for mass spectrometry on day 7 of erythroid maturation, when the cells are predominately basophilic erythroblasts, and on day 12 of erythroid maturation, when they are predominately poly- and ortho- chromatic erythroblasts. The maturation stage of the cells was confirmed by both cytospins and imaging flow cytometric analyses. Two independent replicates were performed and key results confirmed by western blotting. Terminal erythroid maturation was associated with a dramatic decline in the abundance of multiple histone marks associated with active transcription elongation, including Histone H3 lysine 36 di- and tri-methylation (H3K36me2, H3K36me3), and Histone H3 Lysine 79 di-methylation (H3K79me2). Surprisingly, this was not accompanied by an increase in the abundance of repressive heterochromatin marks (H3K27me3, H3K9me3, and H4K20me3) or a global decline in histone acetylation. Histone H4 lysine 16 acetylation (H4K16Ac), associated with RNA polymerase II pause release (Kapoor-Vazirani MCB 2011) significantly declined, but multiple acetylation marks including H3K36Ac and H3K23Ac increased in abundance. As expected, the abundance histone H4 lysine 20 mono-methylation (H4K20me1), which is implicated both in erythroblast chromatin condensation (Malik Cell Reports 2017) and the regulation of RNA Polymerase II pausing (Kapoor-Vazirani MCB 2011) also significantly increased. Consistent with these data, integration of RNA-seq and ChIP-seq data identified 3,058 genes whose expression decreased from basophilic erythroblast to orthochromatic erythroblasts, which lost enrichment for H3K36me3 (mark of active elongation) without accumulating H3K27me3 (heterochromatin mark). Based on these data, we hypothesized that RNA polymerase II pausing is a critical regulator of gene expression in maturing erythroblasts. RNA Polymerase II (Pol II) pausing is a highly regulated mechanism of transcriptional regulation, whereby transcription is initiated, but pauses 30-60bp downstream of the transcription start site. For paused Pol II to be released into active elongation, pTEFb must hyper-phosphorylate Serine 2 of the Pol II c-terminal domain (CTD). Importantly, pTEFb can be directed to specific loci through interaction with transcription factors, including GATA1 (Elagib Blood 2008; Bottardi NAR 2011). Hexim1 is a key regulator of Pol II pausing that sequesters pTEFb and inhibits its action. Consistent with a central role for Pol II pausing dynamics in the regulation of terminal erythroid maturation, Hexim1 is highly expressed in erythroid cells compared to most other cell types and its expression increases during terminal erythroid maturation. Conversely, the expression of CCNT1 and CKD9, the components of pTEFb, decline during terminal maturation, and the level of elongation competent (Ser2 and Ser2/Ser5 CTD phosphorylated) Pol II also decreases dramatically. To gain insights into the function of Pol II pausing in maturing erythroblasts, we induced Hexim1 expression in HUDEP2 cells (Kurita PLoS One 2013) using hexamethane bisacetamide (HMBA). HMBA treatment increased Hexim1 levels a dose dependent manner and was associated with gene expression and phenotypic changes suggestive of accelerated erythroid maturation. Together, these data suggest that RNA Pol II pausing dynamics are an important regulator of terminal erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Description: Key Points There is heterogeneity in the clinical, laboratory, and genetic bases of HX. Alterations in PIEZO1 channel kinetics, response to osmotic stress, and membrane trafficking may contribute to channel dysfunction in HX.