Stefania Bottardi

Ikaros and GATA-1 Combinatorial Effect Is Required for Silencing of Human γ-Globin Genes

ABSTRACT During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human β-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human γ-gene promoters, thereby contributing to γ-globin gene silencing at the time of the γ- to β-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human γ-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.

Ikaros interacts with P-TEFb and cooperates with GATA-1 to enhance transcription elongation

Ikaros is associated with both gene transcriptional activation and repression in lymphocytes. Ikaros acts also as repressor of human γ-globin (huγ-) gene transcription in fetal and adult erythroid cells. Whether and eventually, how Ikaros can function as a transcriptional activator in erythroid cells remains poorly understood. Results presented herein demonstrate that Ikaros is a developmental-specific activator of huγ-gene expression in yolk sac erythroid cells. Molecular analysis in primary cells revealed that Ikaros interacts with Gata-1 and favors Brg1 recruitment to the human β-globin Locus Control Region and the huγ-promoters, supporting long-range chromatin interactions between these regions. Additionally, we demonstrate that Ikaros contributes to transcription initiation and elongation of the huγ-genes, since it is not only required for TBP and RNA Polymerase II (Pol II) assembly at the huγ-promoters but also for conversion of Pol II into the elongation-competent phosphorylated form. In agreement with the latter, we show that Ikaros interacts with Cyclin-dependent kinase 9 (Cdk9), which contributes to efficient transcription elongation by phosphorylating the C-terminal domain of the large subunit of Pol II on Serine 2, and favours Cdk9 recruitment to huγ-promoters. Our results show that Ikaros exerts dual functionality during gene activation, by promoting efficient transcription initiation and elongation.

The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis.

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of IkNULL hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.

Lineage-Specific Transcription Factors in Multipotent Hematopoietic Progenitors: A Little Bit Goes a Long Way

Basal expression of lineage-specific transcription factors (TFs) in multipotent hematopoietic progenitor cells (HPCs) plays a pivotal role in normal hematopoiesis. Indeed, the interplay between lineage-specific TFs and chromatin modifying or remodeling complexes allows chromatin modifications at specific hematopoietic loci and promotes transcriptionally prone conformations. It is now well accepted that during hematopoiesis, the expression of various lineage-specific genes can be preceded by their potentiation i.e., by chromatin activation, in progenitor cells. Gene potentiation appears to counterbalance epigenetic silencing of lineage-specific genes in early progenitors, while maintaining an accessible chromatin conformation in the lineage pathway selected. Herein, we discuss the impact of lineage-specific TFs on gene potentiation and priming in normal hematopoiesis, and emphasize the complementary role of locus control region (LCR) or LCR-like structures and promoter regions in gene-specific potentiation events.

Direct Protein Interactions Are Responsible for Ikaros-GATA and Ikaros-Cdk9 Cooperativeness in Hematopoietic Cells

ABSTRACT Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.

Differential requirement of a distal regulatory region for pre-initiation complex formation at globin gene promoters

Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human β-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the β-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual β-like globin genes. Moreover, in transgenic mice expressing the entire human β-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter β-like globin gene expression. Here, we show that abnormal expression of human β-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human ɛ- and γ-promoters, but not at the β-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human γ-promoters. We also find that HS2 is dispensable for high-level human β-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.

IKAROS: a multifunctional regulator of the polymerase II transcription cycle

Transcription factors are important determinants of lineage specification during hematopoiesis. They favor recruitment of cofactors involved in epigenetic regulation, thereby defining patterns of gene expression in a development- and lineage-specific manner. Additionally, transcription factors can facilitate transcription preinitiation complex (PIC) formation and assembly on chromatin. Interestingly, a few lineage-specific transcription factors, including IKAROS, also regulate transcription elongation. IKAROS is a tumor suppressor frequently inactivated in leukemia and associated with a poor prognosis. It forms a complex with the nucleosome remodeling and deacetylase (NuRD) complex and the positive transcription elongation factor b (P-TEFb), which is required for productive transcription elongation. It has also been reported that IKAROS interacts with factors involved in transcription termination. Here we review these and other recent findings that establish IKAROS as the first transcription factor found to act as a multifunctional regulator of the transcription cycle in hematopoietic cells.