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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 17, 2003 as doi:10.1096/fj.02-0977fje. |
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* Dipartimento di Biologia e Patologia Cellulare e Molecolare c/o Centro di Endocrinologia ed Oncologia Sperimentale del CNR, Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli "Federico II," and
Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania, USA
2Correspondence: Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli "Federico II," via Pansini 5, 80131 Naples, Italy. E-mail: afusco{at}napoli.com
SPECIFIC AIMS
HMGA1 proteins are architectural transcription factors involved in the regulation of chromatin structure and function. Hmga1 gene expression is abundant during embryogenesis and in neoplastic tissues, whereas it is low in adult normal tissues, suggesting a critical role during embryonic development. The aim of this work was to identify the role of HMGA1 proteins during development by in vitro differentiation of wild-type and hmga1-/- embryonic stem (ES) cells.
PRINCIPAL FINDINGS
1. Loss of hmga1 reduces hematopoiesis in ES cell-derived embryoid bodies
To establish the role of HMGA1 proteins during differentiation, we generated hmga1+/- and hmga1-/- embryonic stem (ES) cells. To induce differentiation, ES cells were seeded in methylcellulose-based medium. Under these conditions, they generate tridimensional cystic structures, known as embryoid bodies (EBs), recapitulating embryo development and gene expression. Hmga1-/- ES cells formed fewer and smaller EBs than wild-type and heterozygous ES cells. Hematopoietic differentiation, identified by the appearance of macrophages and granulocytic cells at the edges of the EBs, was reduced by the loss of hmga1 expression. Conversely, erythroid differentiation, detectable as blood islands into the EBs, was not compromised in hmga1 null EBs. FACS and RT-PCR analysis revealed a higher c-kit expression in hmga1-/- EBs compared with wild-type, indicating a more immature cell population in knockout EBs.
2. The T cell differentiation pathway is impaired in hmga1-/- ES cells and embryos
FACS analyses were carried out on EB-derived cells. These experiments showed that the T cell-specific marker Thy-1.2 was expressed at higher levels in wild-type ES cell population (30.3%) than in hmga1 null ES cells (9.3%) (Fig. 1
A), whereas B-220+ cells were more abundant in hmga1 null EBs than in the wild-type (58.7% vs. 29.0%). No difference in expression of the surface marker CD44 was observed between hmga1 +/+ and -/- EBs (Fig. 1A
).
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These data were confirmed by RT-PCR analyses evaluating the expression of pre-B and pre-T cell markers. As shown in Fig. 1B
, Thy-1.2, which was undetectable in undifferentiated ES cells and after 11 days of culture in methylcellulose medium, was expressed after 20 days. According to FACS data, Thy-1.2 expression level was about fivefold lower (as evaluated by densitometric analysis) in hmga1-/- cells than in wild-type ES cells. Reintroduction of hmga1b in ES cells (hmga1-/-R) rescued Thy-1.2 gene expression to levels comparable to wild-type ES cells (Fig. 1B
). This finding indicates that HMGA1 proteins are involved, directly or indirectly, in T cell differentiation. GATA-3, another marker of T cell differentiation, slightly decreased in homozygous knockout cells after 20 days in methylcellulose-based medium (Fig. 1B
) and, similar to Thy-1.2, increased in hmga1-/-R EBs. Conversely, expression of B cell lymphoid markers VpreB and B29 was higher in hmga1 null ES cells than in wild-type cells (Fig. 1B
).
HMGA1 proteins are modulators of the function of many of the transcriptional factors that control cytokine gene expression. Recent findings show that IL-2 and IL-2R expression is essential for the autocrine loop that drives T cell proliferation and clonal expansion after an immune stimulus. To define the mechanisms underlying the reduced capacity of hmga1-/- ES cells to differentiate to T lymphocytes, we analyzed IL-2 expression by RT-PCR and showed that IL-2 was expressed at higher levels in 25-day-old wild-type EBs than in age-matched double knockout EBs (Fig. 1C
). Conversely, expression levels of IL-6, a cytokine that induces terminal maturation of B cells into antibody-producing cells, was higher in hmga1-/- EBs than in wild-type EBs (Fig. 1C
). No changes were observed in IL-7 expression levels (Fig. 1C
).
Undifferentiated hmga1+/-ES cell clones were microinjected into 3.5 day postcoitum (dpc) C57BL/6J blastocysts and strains of hmga1 knockout mice were generated. RT-PCR analyses performed on fetal livers from 14.5 dpc embryos showed that IL-2 expression was higher in wild-type than single and double knockout embryos (Fig. 1C
).
3. Mixed hemopoietic colonies derived from hmga1-/- EBs are reduced in number and size
To identify hemopoietic colonies, 11-day-old EBs were disrupted and the cells were replated in methylcellulose medium. Colonies were observed after 11–20 days. Wild-type ES cells gave rise to erythroid, macrophage, and multilineage colonies. In sharp contrast, hmga1-/- macrophage and multilineage colonies were very scarce whereas erythroid colonies were unaffected.
4. Loss of HMGA1 is associated with increased early megakaryocyte markers and globin gene expression in vitro and in vivo
When megakaryocyte differentiation was induced in ES cells, RT-PCR and cytochemical analyses showed an increase of differentiation in hmga1-/- with respect to wild-type cells. Moreover, expression of megakaryocyte markers was increased in fetal livers from hmga1-/- 14.5 dpc embryos compared with wild-type and single knockout.
To characterize erythropoietic differentiation in hmga1-/- ES cells, globin gene expression was evaluated by RT-PCR in EBs. All four globins analyzed, 
-, 
-, ßH1, and ß major globin genes, were expressed at higher levels in hmga1-/- ES cells than in wild–type, indicating that the hmga1 gene plays a main role in erythroid differentiation. Consistent with the in vitro results, fetal globin (ßH1 and -) gene expression was higher in 9.5 and 14.5 dpc old hmga1-/- yolk sacs and in 14.5 dpc fetal livers than in wild-type tissues.
5. HMGA1 proteins negatively regulate GATA-1 expression in vitro and in vivo by direct binding to its control region
GATA-1 regulates red blood cell differentiation and its consensus sequence is contained in all erythroid-specific genes, including globin genes. Hmga1-/- EBs expressed higher levels of GATA-1 than wild-type and in hmga1-/-R cells GATA-1 expression was reduced. In yolk sacs from 9.5 dpc old embryos, GATA-1 was up-regulated in double knockout compared with wild-type and single knockout embryos, indicating that the expression of hmga1 and GATA-1 is inversely related both in vitro and in vivo.
To investigate the role of HMGA1 on GATA-1 transcription, we transiently transfected wild-type and hmga1-/- ES cells with reporter constructs carrying the luciferase gene under the control of the GATA-1 proximal promoter (IE) with or without the upstream enhancer element (IE3.9intLUC and IE2.6intLUC, respectively). The activity of both constructs was higher in hmga1-/- ES cells than in wild-type cells. When the hmga1-expressing construct was cotransfected with either reporter construct, a sixfold reduction of luciferase activity was observed both in wild-type and double knockout cells. By chromatin immunoprecipitation and electrophoretic mobility shift assays (EMSA), we showed that HMGA1 proteins are able to specifically bind two AT-rich sequences in the GATA-1 upstream activating element. When EMSA analyses were performed using protein extracts from wild-type and hmga1-/- ES cells, hmga1-/- ES cells lacked a specific band. These results indicate that HMGA1 proteins directly down-regulate GATA-1 expression.
CONCLUSIONS AND SIGNIFICANCE
Here we show that HMGA1 proteins are involved in different hematopoietic lineage commitment checkpoints.
HMGA1 proteins regulate lymphoid differentiation
HMGA1 protein expression would force the common lymphoid precursors (CLP) to differentiate to T, rather than B lymphocytes, likely by fine-tuning the expression levels of cytokines regulating B and T cell proliferation/differentiation. T precursor cells were greatly reduced in hmga1-/- ES cells, whereas an increase in the amount of B cell precursors was observed. Reintroduction of the cDNA coding for HMGA1b was able to rescue the wild-type phenotype. From our data, HMGA1 expression would stimulate IL-2 (a T cell inducer) and down-regulate IL-6 (a B cell inducer). Our findings are consistent with previous results showing the central role of HMGA1 proteins in the regulation of IL-2 and other cytokines (Fig. 2
).
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HMGA proteins have a critical role in myeloid differentiation
The impairment of myeloid differentiation observed in hmga1-/- EBs agrees with the finding that HMGA1 proteins physically interact with the ets myeloid transcription factor PU.1 and enhance its transcriptional activity. A reduction in macrophage population was achieved in the hmga1-/- EBs. HMGA1-regulated expression of GM-CSF may account for this result.
HMGA1 proteins affect erythroid/megakaryocytic differentiation
In hmga1-/- EBs, megakaryocytic differentiation, as detected by cytochemical and molecular analyses, and erythroid differentiation, as detected by globin gene expression, were increased despite the general reduction of other hematopoietic colonies. The increased expression of GATA-1 in hmga1 null ES cells may account at least in part for these modifications. In fact, GATA-1 transcription factor is critical for megakaryocyte growth regulation and platelet biogenesis; it regulates erythroid-specific gene expression and affects erythroid differentiation in a dose-dependent manner. Consistently, we showed that HMGA1 proteins are able to specifically bind two AT-rich sequences in the GATA-1 upstream activating element and to down-regulate GATA-1 promoter activity in functional assays.
PU.1 and GATA-1 are essential for the development of myeloid and erythroid lineages, respectively. Each transcription factor functionally inhibits the other. Their overexpression can block differentiation in lineages that normally are down-regulated.
Therefore, we propose a model (Fig. 2)
in which the HMGA1 regulation of hematopoiesis would be mediated by its dual effect on GATA-1 and PU.1.
Our results might represent advancement in understanding the regulation of the lineage commitment in lympho-hematopoietic differentiation, in which HMGA1 proteins surely play a critical role.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0977fje; doi: 10.1096/fj.02-0977fje 
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