ALBERT

Description: The 11-subunit exosome complex (EC) controls the degradation, processing and synthesis of non-coding and coding RNAs. Although EC actions on RNAs can be highly selective, it regulates fundamental biological processes including the DNA damage response, maintenance of genome integrity, stem cell differentiation and erythroid maturation. The EC catalytic subunits Exosc10 and Dis3 degrade RNAs in the nucleus. DIS3 is mutated frequently in human multiple myeloma (Chapman et al., Nature, 2011), although the dysregulated RNA targets are not established. McIver et al. (Blood, 2014, eLife, 2016), demonstrated that EC is an important determinant of erythroid progenitors (BFU-E) and c-Kit signaling. While structural requirements for EC formation and activity are defined, how EC controls processes such as cellular proliferation, survival and differentiation is unclear. During erythropoiesis, the master regulator of erythrocyte development GATA1 represses genes encoding EC subunits. We conducted a multiomic analysis of GATA1-regulated transcripts and proteins in a genetic complementation system (Tanimura et al., Dev. Cell, 2018), which revealed a disproportionately greater loss of EC catalytic subunits Exosc10 and Dis3 (~5 fold), in comparison with other EC subunits, during erythroid maturation. As it is unclear if one or both catalytic subunits are required to generate BFU-E, and EC catalytic subunit functions have not been analyzed in other progenitor contexts, we tested whether the catalytic subunits function similarly or differentially. We conducted loss-of-function studies using shRNAs to downregulate Dis3 or Exosc10 in primary mouse fetal liver cells. Loss of Dis3 mRNA did not impact Exosc10 expression and vice versa. Two shRNAs against Dis3 almost entirely ablated BFU-E, CFU-GM and CFU-GEMM (〉90%, p 〈 0.0001), whereas two shRNAs against Exosc10 decreased BFU-E and CFU-GM by 65 and 55% (p

Description: By functioning as an enzyme cofactor, hemoglobin component and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we used CRISPR/Cas9 to delete intron 1 and 8 GATA motifs in Alas2, encoding the rate-limiting enzyme in heme biosynthesis, in erythroid precursor G1E-ER-GATA-1 cells (Tanimura et al., 2016, EMBO Rep.). These cells stably express a b-estradiol-activated allele encoding the estrogen receptor hormone binding domain fused to GATA-1. The GATA motif mutations abrogated GATA-1-mediated activation of Alas2 and decreased heme ~30 fold. We merged GATA-1/heme-regulated sectors of the proteome and transcriptome. This analysis revealed a GATA-1/heme circuit involving hemoglobin subunits, ubiquitination components, and proteins not implicated in erythrocyte biology, including the zinc exporter Slc30a1. Slc30a1 and the zinc importer Slc39a8 were the most highly expressed zinc transporters in G1E-ER-GATA-1 cells. Both were GATA-1-activated, and Slc30a1, but not Slc39a8, expression declined in heme-deficient Alas2-intronic mutant cells. We tested whether the zinc transporter genes Slc30a1 and Slc39a8 that share GATA-1 regulation and differ in heme regulation are controlled similarly in primary cells. Lineage-depleted hematopoietic precursors from murine fetal livers were cultured for 72 hours, and flow cytometry was used to isolate cells based on erythroid surface markers. Whereas Slc30a1 expression increased 5.7-fold upon differentiation, Slc39a8 increased 4.6-fold during the initial phase, but was downregulated 8.7-fold thereafter. To quantify intracellular zinc levels, we used the fluorescent zinc indicator FluoZin-3. The FluoZin-3 signal increased during early maturation and decreased thereafter, as predicted by the zinc transporter switch, in which expression of the zinc exporter and importer was sustained and decreased, respectively, during terminal differentiation. We demonstrated that the zinc transporter switch occurs in a primary human erythroblast culture system. Intracellular zinc rises during initial erythroid maturation, followed by a steep decline during terminal differentiation. The heme biosynthesis inhibitor succinylacetone reduced SLC30A1 expression 2.8-fold (p 〈 0.01), as well as other heme-dependent globin genes HBB and HBA1. These primary mouse and human cell studies revealed an evolutionarily conserved mechanism to differentially regulate zinc transporter genes during erythroid differentiation, culminating in a switch from importer and exporter expression to solely exporter expression. To test whether reconfiguring the mechanism governing intracellular zinc impacts differentiation and to elucidate Slc30a1 and Slc39a8 function, we developed multiple shRNAs and used them in loss-of-function studies with lineage-negative hematopoietic precursors from murine fetal livers (E14.5). Slc39a8 downregulation decreased intracellular zinc levels in immature erythroblasts. We also used the zinc chelator TPEN to reduce zinc in immature erythroblasts. TPEN reduced intracellular zinc and considerably decreased the live cell population (p 〈 0.001). Downregulating Slc30a1 increased intracellular zinc 2.2-fold and, strikingly, accelerated differentiation (p 〈 0.001). This analysis established a conserved paradigm in which a GATA-1/heme circuit controls trace metal transport machinery and trace metal levels as a mechanism governing cellular differentiation. Ongoing studies involve the development of innovative strategies to leverage this mechanism to enhance or suppress erythrocyte development with primary mouse and human systems. We are conducting multi-disciplinary metallomic analyses to test the hypothesis that the dynamic changes in zinc selectively commission and decommission zinc-binding proteins in erythroblasts. We anticipate that this approach can be readily extended to other sectors of the hematopoietic system and more broadly. Disclosures No relevant conflicts of interest to declare.

Description: New paleomagnetic results from the late Eocene-Middle Miocene samples from Deep Sea Drilling Project Site 274, cored during Leg 28 on the continental rise off Victoria Land, Ross Sea, provide a chronostratigraphic framework for an existing paleoclimate archive during a key period of Antarctic climate and ice sheet evolution. Based on this new age model, the cored late Eocene-Middle Miocene sequence covers an interval of almost 20 Myr (from ∼35 to ∼15 Ma). Biostratigraphic constraints allow a number of possible correlations with the Geomagnetic Polarity Time Scale. Regardless of correlation, average interval sediment accumulation rates above 260 mbsf are ∼6 cm/kyr with the record punctuated by a number of unconformities. Below 260 mbsf (across the Eocene/Oligocene boundary) interval, sedimentation accumulation rates are closer to ∼1 cm/kyr. A major unconformity identified at ∼180 mbsf represents at least 9 Myr accounting for the late Oligocene and Early Miocene and represent non-deposition and/or erosion due to intensification of Antarctic Circumpolar Current activity. Significant fluctuations in grain size and magnetic properties observed above the unconformity at 180 mbsf, in the Early Miocene portion of this sedimentary record, reflect cyclical behavior in glacial advance and retreat from the continent. Similar glacial cyclicity has already been identified in other Miocene sequences recovered in drill cores from the Antarctic margin.