Reciprocal regulation of alternative lineages by Rgs18 and its transcriptional repressor Gfi1b.

Appropriate diversification of cellular lineages from multi-potent progenitors is essential for normal development and homeostasis. The specification of erythroid and megakaryocytic lineages represents an especially vital developmental event whose molecular regulation remains incompletely defined. We now demonstrate the role of Rgs18, a GTPase-activating protein and transcriptional target of the repressor Gfi1b, in regulating these processes in mouse and human cells. Gfi1b stringently represses Rgs18 expression in erythroid cells, whereas, during megakaryocytic differentiation, declining Gfi1b levels facilitate a robust induction of Rgs18. Concordantly, alterations in Rgs18 expression produce disparate outcomes by augmenting megakaryocytic and potently suppressing erythroid differentiation and vice versa. These phenotypes reflect the differential impact of Rgs18 on signaling through p38 MAPK family proteins, and ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively) in the two lineages, which in turn alter the balance between the mutually antagonistic transcription factors Fli1 and Klf1. Overall, these results identify Rgs18 as a new and crucial effector of Gfi1b that regulates downstream signaling and gene expression programs to orchestrate erythro-megakaryocytic lineage choices. This dual role of Rgs18 in reciprocally regulating divergent lineages could exemplify generic mechanisms characteristic of multiple family members in different contexts.

Antagonistic actions of Rcor proteins regulate LSD1 activity and cellular differentiation.

Lysine-specific demethylase 1 (LSD1) demethylates nucleosomal histone H3 lysine 4 (H3K4) residues in collaboration with the corepressor CoREST/REST corepressor 1 (Rcor1) and regulates cell fates by epigenetically repressing gene targets. The balanced regulation of this demethylase, if any, is however unknown. We now demonstrate the actions of two other Rcor paralogs, Rcor2 and Rcor3, in regulating LSD1 enzymatic activity and biological function in hematopoietic cells. All three Rcor proteins interact with LSD1 and with the erythro-megakaryocytic transcription factor growth factor independence (Gfi)1b; however, whereas Rcor2, like Rcor1, facilitates LSD1-mediated nucleosomal demethylation, Rcor3 competitively inhibits this process. Appending the SANT2 domain of Rcor1 to Rcor3 confers the ability to facilitate LSD1-mediated demethylation on the chimeric Rcor protein. Consistent with their biochemical activities, endogenous Rcor1, Rcor2, and LSD1 promote differentiation, whereas Rcor3 opposes these processes. Recruitment of Rcor3 to cognate gene targets by Gfi1b and LSD1 leads to inhibition of H3K4 demethylation of chromatin and transcriptional derepression of these loci. Remarkably, profound alterations in Rcor1/3 levels during erythroid versus megakaryocytic differentiation potentiate antagonistic outcomes. In mature erythroid cells, a strong upsurge in Rcor3 and a sharp decline in Rcor1 levels counteract LSD1/Rcor1/2-mediated differentiation. In contrast, the opposite changes in Rcor1/3 levels in megakaryocytes favor differentiation and likely maintain homeostasis between these lineages. Overall, our results identify Rcor3 as a natural inhibitor of LSD1 and highlight a dual mechanism of regulating the enzymatic activity and restraining the epigenetic impact of this robust demethylase during hematopoietic differentiation.

Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) sustain blood production throughout life. HSCs are capable of extensive proliferative expansion, as a single HSC may reconstitute lethally irradiated hosts. In steady-state, HSCs remain largely quiescent and self-renew at a constant low rate, forestalling their exhaustion during adult life. Whereas nuclear regulatory factors promoting proliferative programmes of HSCs in vivo and ex vivo have been identified, transcription factors restricting their cycling have remained elusive. Here we report that the zinc-finger repressor Gfi-1 (growth factor independent 1), a cooperating oncogene in lymphoid cells, unexpectedly restricts proliferation of HSCs. After loss of Gfi-1, HSCs display elevated proliferation rates as assessed by 5-bromodeoxyuridine incorporation and cell-cycle analysis. Gfi-1-/- HSCs are functionally compromised in competitive repopulation and serial transplantation assays, and are rapidly out-competed in the bone marrow of mouse chimaeras generated with Gfi-1-/- embryonic stem cells. Thus, Gfi-1 is essential to restrict HSC proliferation and to preserve HSC functional integrity.

Cooperative Stimulation of Megakaryocytic Differentiation by Gfi1b Gene Targets Kindlin3 and Talin1.

Understanding the production and differentiation of megakaryocytes from progenitors is crucial for realizing the biology and functions of these vital cells. Previous gene ablation studies demonstrated the essential role of the transcriptional repressor Gfi1b (growth factor independence 1b) in the generation of both erythroid and megakaryocytic cells. However, our recent work has demonstrated the down-regulation of this factor during megakaryocytic differentiation. In this study we identify two new gene targets of Gfi1b, the cytoskeletal proteins Kindlin3 and Talin1, and demonstrate the inverse expression and functions of these cytoskeletal targets relative to Gfi1b, during megakaryocytic differentiation. Both kindlin3 and talin1 promoters exhibit dose dependent Gfi1b and LSD1 (lysine specific demethylase 1; a Gfi1b cofactor) enrichment in megakaryocytes and repression in non-hematopoietic cells. Accordingly the expression of these genes is elevated in gfi1b mutant and LSD1 inhibited hematopoietic cells, while during megakaryocytic differentiation, declining Gfi1b levels fostered the reciprocal upregulation of these cytoskeletal factors. Concordantly, manipulation of Kindlin3 and Talin1 expression demonstrated positive correlation with megakaryocytic differentiation with over-expression stimulating, and inhibition diminishing, this process. Co-operativity between these factors and integrins in promoting differentiation was further underscored by physical interactions between them and integrinß3/CD61 and by stimulation of differentiation by the Talin1 head domain, which is necessary and sufficient for integrin activation. Therefore this study demonstrates the significance of Gfi1b regulated Kindlin3-Talin1 expression in driving megakaryocytic differentiation and highlights the contribution of cytoskeletal agents in the developmental progression of these platelet progenitors.

An unexpected link between notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila.

A fundamental question in hematopoietic development is how multipotent progenitors achieve precise identities, while the progenitors themselves maintain quiescence. In Drosophila melanogaster larvae, multipotent hematopoietic progenitors support the production of three lineages, exhibit quiescence in response to cues from a niche, and from their differentiated progeny. Infection by parasitic wasps alters the course of hematopoiesis. Here we address the role of Notch (N) signaling in lamellocyte differentiation in response to wasp infection. We show that Notch activity is moderately high and ubiquitous in all cells of the lymph gland lobes, with crystal cells exhibiting the highest levels. Wasp infection reduces Notch activity, which results in fewer crystal cells and more lamellocytes. Robust lamellocyte differentiation is induced even in N mutants. Using RNA interference knockdown of N, Serrate, and neuralized (neur), and twin clone analysis of a N null allele, we show that all three genes inhibit lamellocyte differentiation. However, unlike its cell-autonomous function in crystal cell development, Notch's inhibitory influence on lamellocyte differentiation is not cell autonomous. High levels of reactive oxygen species in the lymph gland lobes, but not in the niche, accompany N(RNAi)-induced lamellocyte differentiation and lobe dispersal. Our results define a novel dual role for Notch signaling in maintaining competence for basal hematopoiesis: while crystal cell development is encouraged, lamellocytic fate remains repressed. Repression of Notch signaling in fly hematopoiesis is important for host defense against natural parasitic wasp infections. These findings can serve as a model to understand how reactive oxygen species and Notch signals are integrated and interpreted in vivo.

Differential transcriptional regulation of meis1 by Gfi1b and its co-factors LSD1 and CoREST.

Gfi1b (growth factor independence 1b) is a zinc finger transcription factor essential for development of the erythroid and megakaryocytic lineages. To elucidate the mechanism underlying Gfi1b function, potential downstream transcriptional targets were identified by chromatin immunoprecipitation and expression profiling approaches. The combination of these approaches revealed the oncogene meis1, which encodes a homeobox protein, as a direct and prominent target of Gfi1b. Examination of the meis1 promoter sequence revealed multiple Gfi1/1b consensus binding motifs. Distinct regions of the promoter were occupied by Gfi1b and its cofactors LSD1 and CoREST/Rcor1, in erythroid cells but not in the closely related megakaryocyte lineage. Accordingly, Meis1 was significantly upregulated in LSD1 inhibited erythroid cells, but not in megakaryocytes. This lineage specific upregulation in Meis1 expression was accompanied by a parallel increase in di-methyl histone3 lysine4 levels in the Meis1 promoter in LSD1 inhibited, erythroid cells. Meis1 was also substantially upregulated in gfi1b-/- fetal liver cells along with its transcriptional partners Pbx1 and several Hox messages. Elevated Meis1 message levels persisted in gfi1b mutant fetal liver cells differentiated along the erythroid lineage, relative to wild type. However, cells differentiated along the megakaryocytic lineage, exhibited no difference in Meis1 levels between controls and mutants. Transfection experiments further demonstrated specific repression of meis1 promoter driven reporters by wild type Gfi1b but neither by a SNAG domain mutant nor by a DNA binding deficient one, thus confirming direct functional regulation of this promoter by the Gfi1b transcriptional complex. Overall, our results demonstrate direct yet differential regulation of meis1 transcription by Gfi1b in distinct hematopoietic lineages thus revealing it to be a common, albeit lineage specific, target of both Gfi1b and its paralog Gfi1.

Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1.

Gfi-1 and Gfi-1b are homologous transcriptional repressors involved in diverse developmental contexts, including hematopoiesis and oncogenesis. Transcriptional repression by Gfi proteins requires the conserved SNAG domain. To elucidate the function of Gfi proteins, we purified Gfi-1b complexes and identified interacting proteins. Prominent among these is the corepressor CoREST, the histone demethylase LSD1, and HDACs 1 and 2. CoREST and LSD1 associate with Gfi-1/1b via the SNAG repression domain. Gfi-1b further recruits these cofactors to the majority of target gene promoters in vivo. Inhibition of CoREST and LSD1 perturbs differentiation of erythroid, megakaryocytic, and granulocytic cells as well as primary erythroid progenitors. LSD1 depletion derepresses Gfi targets in lineage-specific patterns, accompanied by enhanced histone 3 lysine 4 methylation at the respective promoters. Overall, we show that chromatin regulatory proteins CoREST and LSD1 mediate transcriptional repression by Gfi proteins. Lineage-restricted deployment of these cofactors through interaction with Gfi proteins controls hematopoietic differentiation.

Changes in replication, nuclear location, and expression of the Igh locus after fusion of a pre-B cell line with a T cell line.

We have previously observed that replication and nuclear location of the murine Igh locus are developmentally regulated during B cell differentiation. In non-B, B, and plasma cells, sequences near the 3' end of the Igh locus replicate early in S while upstream Vh sequences replicate late in S, and the Igh locus is located near the nuclear periphery. In fact, in MEL non-B cells, replication of a 500-kb segment containing Igh-C and flanking sequences occurs progressively later throughout S by 3' to 5' unidirectional fork movement. In contrast, in pro- and pre-B cells, the entire 3-Mb Igh locus is located away from the nuclear periphery and replicates early in S by forks progressing in both directions. In this study, using an 18-81 (pre-B) x BW5147 (T) cell fusion system in which Igh expression is extinguished, we found that in all Igh alleles, Vh sequences replicated later in S than 3' Igh sequences (similar to that detected in BW5147), but the Igh locus was situated away from the nuclear periphery (similar to that observed in 18-81). Thus, pre-B cell-derived Igh genes had changes in replication timing, but not in nuclear location, whereas T cell-derived Igh genes changed their nuclear location but not their replication timing. These data are consistent with the silencing of a pre-B cell-specific replication program in the fusion hybrid cells and independent regulation of the nuclear location of Igh loci.

The zinc-finger proto-oncogene Gfi-1b is essential for development of the erythroid and megakaryocytic lineages.

Gfi-1 and Gfi-1b are novel proto-oncogenes identified by retroviral insertional mutagenesis. By gene targeting, we establish that Gfi-1b is required for the development of two related blood lineages, erythroid and megakaryocytic, in mice. Gfi-1b(-/-) embryonic stem cells fail to contribute to red cells of adult chimeras. Gfi-1b(-/-) embryos exhibit delayed maturation of primitive erythrocytes and subsequently die with failure to produce definitive enucleated erythrocytes. The fetal liver of mutant mice contains erythroid and megakaryocytic precursors arrested in their development. Myelopoiesis is normal. Therefore, Gfi-1b is an essential transcriptional regulator of erythroid and megakaryocyte development.

Yin Yang 1 is a lipopolysaccharide-inducible activator of the murine 3' Igh enhancer, hs3.

The 3' Igh enhancers, DNase I hypersensitive site (hs) 3B and/or hs4, are required for germline transcription, and hence, class switch recombination for multiple isotypes. A number of hs3-binding transcription factors have been identified by EMSA, including octamer and NF-kappa B family members, and Pax5. We have found that the binding of the transcription factor, Yin Yang 1 (YY1), to hs3 and to the mu E1 site of the intronic enhancer, E mu, is induced in primary splenic B cells after approximately 48 h in response to LPS and other activators of class switch recombination. Transient transfection experiments in B cell lines indicate that YY1 is an activator of hs3. Interestingly, levels of YY1 expression are unchanged in resting and LPS-stimulated B cells. Mixing experiments followed by EMSA showed that a protein present in resting B cells prevented binding of YY1 to DNA. We found that recombinant retinoblastoma protein (Rb) inhibited binding of YY1 to hs3 in a dose-dependent manner, and we have identified complexes of endogenous YY1 with the Rb in resting B cells, but not in LPS-stimulated B cells. A difference in Rb phosphorylation state was also confirmed between resting (G(0)) B cells and LPS-stimulated B cells. These observations suggest that the interaction of YY1 with hypophosphorylated Rb in resting B cells prevents interaction of YY1 with DNA. After stimulation with class-switching activators, such as LPS, Rb becomes hyperphosphorylated and YY1 is released and can then bind to the hs3 enhancer and E mu.