Bcl11a Deficiency Leads to Hematopoietic Stem Cell Defects with an Aging-like Phenotype

SummaryB cell CLL/lymphoma 11A (BCL11A) is a transcription factor and regulator of hemoglobin switching that has emerged as a promising therapeutic target for sickle cell disease and thalassemia. In the hematopoietic system, BCL11A is required for B lymphopoiesis, yet its role in other hematopoietic cells, especially hematopoietic stem cells (HSCs) remains elusive. The extensive expression of BCL11A in hematopoiesis implicates context-dependent roles, highlighting the importance of fully characterizing its function as part of ongoing efforts for stem cell therapy and regenerative medicine. Here, we demonstrate that BCL11A is indispensable for normal HSC function. Bcl11a deficiency results in HSC defects, typically observed in the aging hematopoietic system. We find that downregulation of cyclin-dependent kinase 6 (Cdk6), and the ensuing cell-cycle delay, correlate with HSC dysfunction. Our studies define a mechanism for BCL11A in regulation of HSC function and have important implications for the design of therapeutic approaches to targeting BCL11A

Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation

Here, we describe that lysine-specific demethylase 1 (Lsd1/KDM1a), which demethylates histone H3 on Lys4 or Lys9 (H3K4/K9), is an indispensible epigenetic governor of hematopoietic differentiation. Integrative genomic analysis, combining global occupancy of Lsd1, genome-wide analysis of its substrates H3K4 monomethylation and dimethylation, and gene expression profiling, reveals that Lsd1 represses hematopoietic stem and progenitor cell (HSPC) gene expression programs during hematopoietic differentiation. We found that Lsd1 acts at transcription start sites, as well as enhancer regions. Loss of Lsd1 was associated with increased H3K4me1 and H3K4me2 methylation on HSPC genes and gene derepression. Failure to fully silence HSPC genes compromised differentiation of hematopoietic stem cells as well as mature blood cell lineages. Collectively, our data indicate that Lsd1-mediated concurrent repression of enhancer and promoter activity of stem and progenitor cell genes is a pivotal epigenetic mechanism required for proper hematopoietic maturation. DOI: http://dx.doi.org/10.7554/eLife.00633.00

FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors

During haematopoiesis, megakaryocytes and erythrocytes derive from a common precursor called preMegE. This study reports a role for the transcription factor FOG-1 in specification of preMegEs, while GATA-1 is subsequently required for erythroid-lineage commitment

Generation of bivalent chromatin domains during cell fate decisions

<p>Abstract</p> <p>Background</p> <p>In self-renewing, pluripotent cells, bivalent chromatin modification is thought to silence (H3K27me3) lineage control genes while 'poising' (H3K4me3) them for subsequent activation during differentiation, implying an important role for epigenetic modification in directing cell fate decisions. However, rather than representing an equivalently balanced epigenetic mark, the patterns and levels of histone modifications at bivalent genes can vary widely and the criteria for identifying this chromatin signature are poorly defined.</p> <p>Results</p> <p>Here, we initially show how chromatin status alters during lineage commitment and differentiation at a single well characterised bivalent locus. In addition we have determined how chromatin modifications at this locus change with gene expression in both ensemble and single cell analyses. We also show, on a global scale, how mRNA expression may be reflected in the ratio of H3K4me3/H3K27me3.</p> <p>Conclusions</p> <p>While truly 'poised' bivalently modified genes may exist, the original hypothesis that all bivalent genes are epigenetically premarked for subsequent expression might be oversimplistic. In fact, from the data presented in the present work, it is equally possible that many genes that appear to be bivalent in pluripotent and multipotent cells may simply be stochastically expressed at low levels in the process of multilineage priming. Although both situations could be considered to be forms of 'poising', the underlying mechanisms and the associated implications are clearly different.</p

The earliest thymic T cell progenitors sustain B cell and myeloid lineage potential

The stepwise commitment from hematopoietic stem cells in the bone marrow to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage-restricted progenitors. However, the commitment stage at which progenitors migrate from the bone marrow to the thymus remains unclear. Here we provide functional and molecular evidence at the single-cell level that the earliest progenitors in the neonatal thymus had combined granulocyte-monocyte, T lymphocyte and B lymphocyte lineage potential but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of candidate thymus-seeding progenitors in the bone marrow, which were closely related at the molecular level. Our findings establish the distinct lineage-restriction stage at which the T cell lineage-commitment process transits from the bone marrow to the remote thymus. © 2012 Nature America, Inc. All rights reserved

Initial seeding of the embryonic thymus by immune-restricted lympho-myeloid progenitors

The final stages of restriction to the T cell lineage occur in the thymus after the entry of thymus-seeding progenitors (TSPs). The identity and lineage potential of TSPs remains unclear. Because the first embryonic TSPs enter a non-vascularized thymic rudiment, we were able to directly image and establish the functional and molecular properties of embryonic thymopoiesis-initiating progenitors (T-IPs) before their entry into the thymus and activation of Notch signaling. T-IPs did not include multipotent stem cells or molecular evidence of T cell-restricted progenitors. Instead, single-cell molecular and functional analysis demonstrated that most fetal T-IPs expressed genes of and had the potential to develop into lymphoid as well as myeloid components of the immune system. Moreover, studies of embryos deficient in the transcriptional regulator RBPJ demonstrated that canonical Notch signaling was not involved in pre-thymic restriction to the T cell lineage or the migration of T-IPs

Lineage commitment to a T

he hematopoietic development is a highly dynamic but tightly regulated process. The flexibility to produce blood cells through more than one route will allow the blood system to respond rapidly in stress situations such as infections. On the other hand, regulation of the blood system is required to prevent blood cells from uncontrolled proliferation that could result in leukemia or bone marrow failure due to depletion of blood cells. Hematopoietic stem cells are cells that can differentiate to all blood lineages and maintain the whole hematopoietic system throughout a lifetime. This differentiation process through which stem cells generate mature blood cells occurs through different lineage commitment steps. The precise mapping of the lineage commitment events in normal hematopoietic development is essential to understand its regulation and also to unravel the underlying mechanisms in various hematological diseases. The aim of my thesis has been to study and delineate early lineage commitment processes in the hematopoietic stem cell compartment and in lymphopoiesis with a particular emphasis on T cell development in adult and fetal hematopoiesis using the mouse as a model system. In the thesis I present studies that support an alternative lineage commitment model to the classical and prevailing one for hematopoietic development. In the alternative model hematopoietic stem cells generate progenitors with lymphoid/myeloid (granulocyte/monocyte) and megakaryocyte/erythrocyte/myeloid fates as a first step in their lineage commitment. As the progenitors go through the differentiation to become T cells, they do not commit to a final T cell fate until they have reached and entered their final destination, the thymus. In my studies we demonstrate that the progenitor that initially seeds the thymus during early embryonic development and replenishes the thymus postnatally is more multipotent than previously appreciated. This model has not only identified new and critical commitment steps but also provides a better candidate target cell for the origin of biphenotypic leukemia, which is characterized by lymphoid and myeloid phenotypes

Delineating the cellular pathways of hematopoietic lineage commitment.

The prevailing model for adult hematopoiesis postulates that the first lineage commitment step results in a strict separation of common myeloid and common lymphoid pathways. However, the recent identification of granulocyte/monocyte (GM)-lymphoid restricted lymphoid-primed multipotent progenitors (LMPPs) and primitive common myeloid progenitors (CMPs) within the "HSC" compartment provide compelling support for establishment of independent GM-megakaryocyte/erythroid (GM-MkE) and GM-lymphoid commitment pathways as decisive early lineage fate decisions. These changes in lineage potentials are corroborated by corresponding changes in multilineage transcriptional priming, as LMPPs down-regulate MkE priming but become GM-lymphoid transcriptionally primed, whereas CMPs are GM-MkE primed. These distinct biological and molecular relationships are established already in the fetal liver

T-RHEX-RNAseq – a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells

Abstract Background RNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming – to allow analysis of a broader range of transcripts – remains challenging. Results We here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps. Conclusion T-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells

Additional file 1 of T-RHEX-RNAseq – a tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells

Additional file 1: Figure S1. Schematic overview of the T-RHEX-RNAseq protocol outlining adapters, adapter introduction and primers used to amplify the library. In brief, double stranded cDNA reverse transcription with dUTP incorporated during the second strand synthesis is subjected to tagmentation with Tn5 loaded with i5 adapters. The i7 adapters are introduced by annealing an i7 oligo to the covalently attached part of the i5 adapter. Subsequently, gap fill in combination with ligation is used to covalently attach the i7 adapter. As Phusion is unable to utilize the dUTP containing strand as a template, stranded libraries are then generated by amplification using Pr2 in combination with the i5 completion primer. Figure S2. Strand-specificity of Tn-RNAseq and Directional Tn-RNAseq libraries. (A). Percentage of reads in exons: localized in a matched or mismatched orientation to transcript; or alternatively being localized in regions with overlapping antiparallel transcripts (undetermined). The data from Gertz et al., [1] was downloaded and processed using nf-core and strand-specificity evaluated using RSeq QC. Figure S3. Tracks and duplication rates of T-RHEX-RNAseq libraries from primary hematopoietic stem- and progenitor cells. (A) Tracks showing plus and minus strand reads in the Neat1, Kit, Hspd1 and Hspe1 genomic regions in primary mouse hematopoietic stem cells (HSCs) and lymphoid primed multipotent progenitors (LMPPs). Arrows below the gene names indicate the 5’-3’ direction of the transcript. RNAseq libraries were prepared directly from the indicated numbers of cells lysed in Single cell lysis solution (SCLS). The use of rRNA blocking reagents and dilution of the blocking reagent is indicated in parenthesis. (B) Reoccurrence (duplication rates) of reads in the indicated libraries. Figure S4. T-RHEX-RNAseq provides highly reproducible data. Spearman correlation between rlog of gene expression in samples generated from the indicated population. The use of rRNA blocking reagents and dilution of the blocking reagent is indicated in parenthesis. Hematopoietic stem cell (HSC with or without CD49b expression); Multipotent progenitor (MPP with no or low CD150 expression), lymphoid primed multipotent progenitors (LMPPs); granulocyte/monocyte progenitors (GMP); and antigen specific CD4 T cells (T, from wild-type or Bhlhe40 knockout mice). Data is from proof-of-principle experiments (HSC and LMPP; 250 and 500 cells respectively) or the subsequently generated T-RHEX-RNAseq data from antigen specific CD4 T cells (1000 cells) [1] and hematopoietic stem/progenitor cells (HSPCs; 250-500 cells) [2]. Table S1. Sample metrics and QC. Supplemental working protocol