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Epigenetic plasticity of erythroid progenitors

Approaches to hepatocyte rescue may be needed in individuals who no longer express the transgene because of concomitant drug toxicity, nonalcoholic steatohepatitis, or viral cirrhosis, which is not uncommon in the hemophilia population. Efforts to develop steroid-sparing immunosuppression or vector decoys to reduce the host anti-AAV capsid T-cell response that limits gene expression is critical. Finally, careful surveillance is important for determining long-term risks, such as AAV integration and hepatocellular cancer (HCC), given the continuing risk for HCC in those with hemophilia (see figure).

Although these are encouraging findings, much work remains. Novel approaches are needed to adapt current vector-gene technology for those excluded from gene therapy: those with inhibitor alloantibodies, natural AAV antibody, 6 and active hepatitis B or hepatitis C liver disease.
Approaches to hepatocyte rescue may be needed in individuals who no longer express the transgene because of concomitant drug toxicity, nonalcoholic steatohepatitis, or viral cirrhosis, which is not uncommon in the hemophilia population. 7 Efforts to develop steroid-sparing immunosuppression or vector decoys to reduce the host anti-AAV capsid T-cell response that limits gene expression 8 is critical. Finally, careful surveillance is important for determining long-term risks, such as AAV integration and hepatocellular cancer (HCC), 9 given the continuing risk for HCC in those with hemophilia 10 (see figure).
In summary, it is exciting to consider that after years of complications of hemophilia factor therapy, including inhibitor formation, hepatitis, and HIV, it is now possible to receive a single dose of gene therapy and achieve therapeutic factor levels sufficient to prevent bleeding episodes and avoid factor use and its complications, while providing hope for those globally affected and achieving an improved quality of life. Epigenetic plasticity of erythroid progenitors

Marjorie Brand | Ottawa Hospital Research Institute
In this issue of Blood, Yu et al, 1 report unexpected cell fate plasticity in erythroid progenitors (EPs) whereby removal or inhibition of the chromatin modifier LSD1 (also called KDM1a) triggers lineage conversion toward a myeloid fate, both in vitro and in vivo. This finding is important because it shows that progenitors that are typically thought of as being committed to the erythroid lineage have in fact retained the potential to differentiate toward other hematopoietic lineages. Furthermore, these findings provide strong support for the idea that active lineage restriction through epigenetic mechanisms, rather than irreversible loss of cell fate potential, underlies cell differentiation in hematopoiesis.
The search for strategies to reactivate fetal g-globin genes expression in adult erythroid cells dates back to the discovery that even a small increase in the level of fetal hemoglobin is enough to significantly alleviate symptoms of b-globinopathies, including sickle cell disease and b-thalassemia. 2 This early finding set the stage for decades-long research that led to the identification of multiple factors that inhibit fetal g-globin genes expression, including transcriptional repressors (eg, BCL11A 3 ) and chromatinmodifying enzymes. Although recent gene therapy trials targeting BCL11A have shown great promise for patients with sickle cell disease or b-thalassemia, 4,5 the development of pharmacological agents to reactivate fetal hemoglobin remains a priority. A promising target for pharmacological induction of fetal hemoglobin is LSD1, an epigenetic enzyme that interacts with BCL11A and represses g-globin genes transcription through the removal of active histone marks H3K4me1 and H3K4me2. 6 Although previous studies showed that LSD1 inhibitors increase fetal hemoglobin levels in animal models, 7 these chemicals also partly block erythroid differentiation, and the effects of prolonged loss of LSD1 activity in erythroid cells remain unknown.
To address this question, Yu  were fluorescently marked with tandem tomato (TdT). Using this approach, they observed that GMP-like cells that arise in vivo upon LSD1 knockout are TdT 1 , which confirms that those cells originate from EPs. Importantly, they also showed that erythroid-derived GMP-like cells give rise to mature myeloid cells, which further supports the idea of an erythroidto-myeloid switch upon LSD1 ablation. Further supporting this observation, they detected a reversal of the ratio between lineage-specifying transcription factors that control the erythroid (eg, GATA1) vs myeloid (eg, PU.1) cell fate transition and found increased binding of the myeloid transcription factor RUNX1 to the Pu.1 gene accompanied by enhanced H3K4me2 signal. Finally, they provided evidence that the erythroid-tomyeloid lineage conversion can be recapitulated in vitro using purified CFU-e cells exposed to an LSD1 inhibitor, which confirms that LSD1 acts through its enzymatic activity to restrict lineage potential in EPs (see figure). Going forward, it is important to establish the full extent of chromatin changes (eg, H3K4me 1/2) and changes in transcription factors binding that occur upon LSD1 inhibition. Furthermore, the possibility that LSD1's function in maintaining erythroid identity is also mediated through demethylation of nonhistone proteins remains to be addressed. Finally, the observation of cell fate plasticity upon LSD1 inhibition raises the possibility of increased susceptibility to malignant transformation by spurious activation of oncogenes, either through stochastic changes in gene expression or in response to environmental fluctuations. Regardless of the mechanism, malignant transformation should be carefully monitored when using pharmacological agents that can potentially alter cell fate decisions in the context of b-globinopathies or other diseases. Previous studies have shown that during erythropoiesis, RNA polymerase II (Pol II) transcription becomes more and more restricted to genes essential for the function of red blood cells. 2 This is thought to be primarily the result of an overall decrease in chromatin accessibility and restricted recruitment of Pol II to erythroid genes by cell-type-specific transcription factors, including GATA1. Murphy et al demonstrate that although histone marks associated with active transcription (eg, H3 lysine 36 di-and trimethylation [H3K36me2/3] and H3 lysine 79 di-methylation [H3K79me2]) decrease, heterochromatin-associated histone modifications (eg, H3 lysine 27 di-and tri-methylation [H3K27me2/3] and histone H3 lysine 9 di-and trimethylation [H3K9me2/3]) do not change during maturation of erythroid cells (see figure). Moreover, the authors demonstrate that elongation competent Pol II becomes limited during differentiation, which leads to an increased pausing index at genes not essential for the development of red blood cells. Erythroid-specific genes maintain efficient recruitment of elongation competent transcription complexes despite the marked reduction in Pol II. This study highlights an as yet underappreciated aspect of transcription regulation during erythropoiesis: the repression of transcription elongation at nonerythroid genes during a transient phase of erythroid maturation.
The authors used primary human CD34 1 hematopoietic progenitor cells and induced differentiation along the erythroid lineage. At day 7, representing intermediate erythroblasts, and at day 10, representing mature erythroblasts, the cells were analyzed by mass spectrometry focusing on histone PTMs. The data show that histone PTMs associated with Pol II transcription elongation declined whereas PTMs typically associated with heterochromatin remained unchanged. ChIPseq, ATAC-seq, and Cut and Tag experiments verified that decline in transcription activity was not associated with heterochromatin formation. Instead, the authors found that the decline in transcription of nonerythroid genes was the result of a defect in transcription elongation (at day 7) and eventually was the result of the lack of recruitment of Pol II (day 10). Remarkably, erythroid genes maintained high levels of transcribing Pol II. The finding that intermediate erythroblasts repress nonerythroid genes at the level of transcription elongation rather than at the level of chromatin accessibility or Pol II recruitment is unexpected and suggests a novel mechanism involved in erythroid cell specification. Intriguingly, the levels of activities that inhibit transcription elongation factor P-TEFb, HEXIM1, and the noncoding RNA 7SK, increase during erythroid maturation, with HEXIM1 exhibiting the highest levels in intermediate erythroblasts, at which stage the Pol II pausing index at nonerythroid genes is increased. The levels of P-TEFb decrease during erythropoiesis. Murphy et al also show that overexpression of HEXIM1 or HEXIM1 haploinsufficiency causes erythroid differentiation defects.
How do P-TEFb and HEXIM1 fit into the process of cell-specific gene regulation during erythropoiesis? After transcription initiation, Pol II pauses to allow capping of the 5' end of the RNA. 3 This pause is mediated by negative elongation factors. P-TEFb phosphorylates these negative elongation factors as well as the Pol II C-terminal domain at serine 2, leading to the formation of an elongation competent transcription complex. How the reduced pool of transcriptionally active P-TEFb and elongation competent Pol II is preferentially recruited to erythroid genes becomes an important question, as emphasized by the Murphy et al study. P-TEFb consists of the CDK9 kinase and cyclin T1 or T2. 4 HEXIM1 and 7SK RNA interact with the cyclin T1 subunit of P-TEFb. This renders P-TEFb unable to function as a transcription elongation factor. Several transcription factors have been shown to interact with cyclin T1 thereby dissociating HEXIM1 and allowing P-TEFb to activate transcription. 4 For example, the HIV-encoded Tat protein efficiently competes with HEXIM1 for interactions with cyclin T and recruits active P-TEFb to paused Pol II in the HIV genome. 5 With respect to hematopoietic cells, previous studies have shown that GATA1 and Ikaros transcription factors recruit P-TEFb to megakaryocytic and erythroid genes. [6][7][8] This is consistent with the study by Murphy et al, which