Enhancer and Transcription Factor Dynamics during Myeloid Differentiation Reveal an Early Differentiation Block in <i>Cebpa null</i> Progenitors

Transcription factors PU.1 and CEBPA are required for the proper coordination of enhancer activity during granulocytic-monocytic (GM) lineage differentiation to form myeloid cells. However, precisely how these factors control the chronology of enhancer establishment during differentiation is not known. Through integrated analyses of enhancer dynamics, transcription factor binding, and proximal gene expression during successive stages of murine GM-lineage differentiation, we unravel the distinct kinetics by which PU.1 and CEBPA coordinate GM enhancer activity. We find no evidence of a pioneering function of PU.1 during late GM-lineage differentiation. Instead, we delineate a set of enhancers that gain accessibility in a CEBPA-dependent manner, suggesting a pioneering function of CEBPA. Analyses of Cebpa null bone marrow demonstrate that CEBPA controls PU.1 levels and, unexpectedly, that the loss of CEBPA results in an early differentiation block. Taken together, our data provide insights into how PU.1 and CEBPA functionally interact to drive GM-lineage differentiation

C/EBPα Is Dispensable for the Ontogeny of PD-1+ CD4+ Memory T Cells but Restricts Their Expansion in an Age-Dependent Manner

<div><p>Ageing and cancer is often associated with altered T cell distributions and this phenomenon has been suggested to be the main driver in the development of immunosenescence. Memory phenotype PD-1+ CD4+ T cells accumulate with age and during leukemic development, and they might account for the attenuated T cell response in elderly or diseased individuals. The transcription factor C/EBPα has been suggested to be responsible for the accumulation as well as for the senescent features of these cells including impaired TCR signaling and decreased proliferation. Thus modulating the activity of C/EBPα could potentially target PD-1+ CD4+ T cells and consequently, impede the development of immunosenescence. To exploit this possibility we tested the importance of C/EBPα for the development of age-dependent PD-1+ CD4+ T cells as well as its role in the accumulation of PD-1+ CD4+ T cells during leukemic progression. In contrast to earlier suggestions, we find that loss of C/EBPα expression in the lymphoid compartment led to an increase of PD-1+ CD4+ T cells specifically in old mice, suggesting that C/EBPα repress the accumulation of these cells in elderly by inhibiting their proliferation. Furthermore, C/EBPα-deficiency in the lymphoid compartment had no effect on leukemic development and did not affect the accumulation of PD-1+ CD4+ T cells. Thus, in addition to contradict earlier suggestions of a role for C/EBPα in immunosenescence, these findings efficiently discard the potential of using C/EBPα as a target for the alleviation of ageing/cancer-associated immunosenescence.</p></div

Increase in PD-1+ CD4+ T cells during ageing and in development of AML.

<p>(A) Spleen cells from 2 months old and 14 months old mice were stained with antibodies against CD4 and PD-1. (B) Quantification of the data in (A) is presented as mean +/− SD, (young: n = 3, old: n = 7). (C) PD-1- CD4+ and PD-1+ CD4+ splenic T cells from 14 months old mice were analyzed for expression of <i>Cebpa</i> normalized to <i>β-actin</i> by qRT-PCR. Data are presented as mean +/− SEM, (n = 7). (D) Spleens from 3 months old mice were stained for CD4, PD-1, CD44 and CD62L. A representative example is shown (n = 5). (E) The spleens from healthy (age-matched, non-transplanted) and leukemic mice were analyzed for PD-1+ CD4+ T cells. **P<0.01; n.s.: not significant.</p

C/EBPα is dispensable for the differentiation of lymphoid cells in young mice.

<p>(A and B) Analysis of DN1-DN4 T cells and (C and D) CD4+ and/or CD8+ T cells in thymi from 2 months old <i>Cebpa</i>^fl/fl (n = 4) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 5) mice. (E and F) Analysis of mature hematopoietic lineages in spleens from 2 months old <i>Cebpa</i>^fl/fl (n = 3) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 4) mice. (G and H) Analysis of the mature hematopoietic lineages in BMs from 2 months old <i>Cebpa</i>^fl/fl (n = 5) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 6) mice. (I and J) Analysis of the PD-1+ CD4+ T cells in spleens from 2 months old <i>Cebpa</i>^fl/fl (n = 3) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 4) mice. (K and L) Analysis of the PD-1+ CD4+ T cells in BMs from 2 months old <i>Cebpa</i>^fl/fl (n = 5) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 6) mice. (M–O) Analysis of CD44 and CD62L subsets within PD-1- and PD1+ CD4+ T cells in spleens from 3 months old <i>Cebpa</i>^fl/fl (n = 5) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 5) mice. The contour plots are examples from <i>Cebpa</i>^fl/fl mice. Mean +/− SD; n.s. = not significant.</p

C/EBPα restricts the formation of PD-1+ CD4+ T cells in spleens of old mice.

<p>(A and B) Analysis of PD-1+ CD4+ T cells in spleens from 14 months old <i>Cebpa</i>^fl/fl (n = 7) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 8) mice. (C and D) Analysis of mature hematopoietic lineages in spleens from 14 months old <i>Cebpa</i>^fl/fl (n = 7) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 8) mice. (E and F) Analysis of the PD-1+ CD4+ T cells as well as the mature hematopoietic lineages (G and H) in BMs from 14 months old <i>Cebpa</i>^fl/fl (n = 7) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 8) mice. The contour plots are examples from <i>Cebpa</i>^fl/fl mice. Mean +/− SD; *P<0.05; **P<0.01; n.s.: not significant.</p

C/EBPα inhibits proliferation of CD4+ T cells in old mice.

<p>(A and B) Sorted PD-1- CD4+ and PD-1+ CD4+ T cells from 2 months old <i>Cebpa</i>^fl/fl and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> mice were assessed for transcripts for the indicated genes by qRT-PCR. The relative expression were normalized to β-actin and presented as mean of <i>Cebpa</i>^fl/fl n = 7 and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> n = 8+/− SEM. (C) CFSE labeled splenocytes from 2 months old <i>Cebpa</i>^fl/fl and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> mice were cultured with or without CD3 and CD28 antibodies and after 72 hours after the splenocytes were stained with CD4 antibody and assayed by flow cytometry. Black and grey lines indicate non-stimulated and stimulated cells, respectively. The numbers of cell divisions as given by the Proliferation feature of FlowJo are shown. (D) Quantification of CD4+ T cells in cell cycle 0–4. (<i>Cebpa</i>^fl/fl n = 3, <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> n = 3). (E and F) Analysis of proliferation of CD4+ T cells in the spleen of 10 to 15 months old <i>Cebpa</i>^fl/fl (n = 8) and <i>Cebpa</i>^fl/fl;<i>CD2iCre</i> (n = 12) mice. The contour plot and histograms are examples from <i>Cebpa</i>^fl/fl mice. Mean +/− SD; *P<0.05; **P<0.01; n.s.: not significant.</p

Initiation of MLL-rearranged AML is dependent on C/EBPα

MLL-fusion proteins are potent inducers of oncogenic transformation, and their expression is considered to be the main oncogenic driving force in ∼10% of human acute myeloid leukemia (AML) patients. These oncogenic fusion proteins are responsible for the initiation of a downstream transcriptional program leading to the expression of factors such as MEIS1 and HOXA9, which in turn can replace MLL-fusion proteins in overexpression experiments. To what extent MLL fusion proteins act on their own during tumor initiation, or if they collaborate with other transcriptional regulators, is unclear. Here, we have compared gene expression profiles from human MLL-rearranged AML to normal progenitors and identified the myeloid tumor suppressor C/EBPα as a putative collaborator in MLL-rearranged AML. Interestingly, we find that deletion of Cebpa rendered murine hematopoietic progenitors completely resistant to MLL-ENL–induced leukemic transformation, whereas C/EBPα was dispensable in already established AMLs. Furthermore, we show that Cebpa-deficient granulocytic-monocytic progenitors were equally resistant to transformation and that C/EBPα collaborates with MLL-ENL in the induction of a transcriptional program, which is also apparent in human AML. Thus, our studies demonstrate a key role of C/EBPα in MLL fusion–driven transformation and find that it sharply demarcates tumor initiation and maintenance

The Proline-Histidine-Rich CDK2/CDK4 Interaction Region of C/EBPα Is Dispensable for C/EBPα-Mediated Growth Regulation In Vivo

The C/EBPα transcription factor regulates growth and differentiation of several tissues during embryonic development. Several hypotheses as to how C/EBPα inhibits cellular growth in vivo have been derived, mainly from studies of tissue culture cells. In fetal liver it has been proposed that a short, centrally located, 15-amino-acid proline-histidine-rich region (PHR) of C/EBPα is responsible for the growth-inhibitory function of the protein through its ability to interact with CDK2 and CDK4, thereby inhibiting their activities. Homozygous Cebpa(ΔPHR/ΔPHR) (ΔPHR) mice, carrying a modified cebpa allele lacking amino acids 180 to 194, were born at the Mendelian ratio, reached adulthood, and displayed no apparent adverse phenotypes. When fetal livers from the ΔPHR mice were analyzed for their expression of cell cycle markers, bromodeoxyuridine incorporation, cyclin-dependent kinase 2 kinase activity, and global gene expression, we failed to detect any cell cycle or developmental differences between the ΔPHR mice and their control littermates. These in vivo data demonstrate that any C/EBPα-mediated growth repression via the PHR as well as the basic region is dispensable for proper embryonic development of, and cell cycle control in, the liver. Surprisingly, control experiments performed in C/EBPα null fetal livers yielded similar results

Differences in Cell Cycle Status Underlie Transcriptional Heterogeneity in the HSC Compartment

Summary: Hematopoietic stem cells (HSCs) are considered a heterogeneous cell population. To further resolve the HSC compartment, we characterized a retinoic acid (RA) reporter mouse line. Sub-fractionation of the HSC compartment in RA-CFP reporter mice demonstrated that RA-CFP-dim HSCs were largely non-proliferative and displayed superior engraftment potential in comparison with RA-CFP-bright HSCs. Gene expression analysis demonstrated higher expression of RA-target genes in RA-CFP-dim HSCs, in contrast to the RA-CFP reporter expression, but both RA-CFP-dim and RA-CFP-bright HSCs responded efficiently to RA in vitro. Single-cell RNA sequencing (RNA-seq) of >1,200 HSCs showed that differences in cell cycle activity constituted the main driver of transcriptional heterogeneity in HSCs. Moreover, further analysis of the single-cell RNA-seq data revealed that stochastic low-level expression of distinct lineage-affiliated transcriptional programs is a common feature of HSCs. Collectively, this work demonstrates the utility of the RA-CFP reporter line as a tool for the isolation of superior HSCs. : HSCs are considered a functional heterogeneous population. Lauridsen et al. use scRNA-seq to demonstrate that most transcriptional heterogeneity within the HSC compartment is associated with differences in cell cycle status. They further use an RA-CFP reporter mouse line to isolate slow-cycling HSCs characterized by superior engraftment potential. Keywords: hematopoietic stem cells, single-cell RNA-sequencing, retinoic acid, transcriptional heterogeneity, hematopoiesi