Predicted structure and selectivity of 3d transition metal complexes with glutamic N , N -bis(carboxymethyl) acid

International audienceStructure and selectivity of 3d transition metal complexes with glutamic N , N -bis(carboxymethyl) acid are analyzed and predicted from DFT calculations

The IKAROS Interaction with a Complex Including Chromatin Remodeling and Transcription Elongation Activities Is Required for Hematopoiesis

<div><p>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 Ik^NULL 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.</p></div

Model of IKAROS dosage effect for formation and recruitment of the NuRD-P-TEFb complex and transcription elongation control of IKAROS-target genes.

<p><i>Productive elongation</i>: when expressed at high levels, as in lineage negative hematopoietic progenitor cells, IKAROS (Ik) associates Mi2/NuRD and CDK9/P-TEFb activities into one complex at gene transcription start site (TSS) and throughout the ORF of IKAROS-target genes. IKAROS dependent enrichment of PP1α at TSS provides a control to P-TEFb activation by the dephosphorylation of CDK9. Once activated, CDK9/P-TEFb phosphorylates Ser-2 on POL II CTD as well as NELF and DSIF, two negative regulators of POL II elongation and thereby, releases the promoter-proximal paused POL II. Then, IKAROS and the NuRD-P-TEFb complex assist POL II during elongation. Based on results obtained by LC-MS/MS analysis, the latter might occurs along with SEC subunits. <i>Paused transcription</i>: the gradual decrease of IKAROS protein concentration, such as normally observed at defined stages of hematopoietic cell differentiation, still allows Mi2/NuRD loading at gene regulatory regions as well as PIC formation at gene promoters. However, lack of P-TEFb and PP1α recruitment results in promoter-proximal pausing of POL II. <i>Gene silencing</i>: in absence of IKAROS, as observed in lymphoma and leukemia clones carrying mutated and/or deleted alleles of IKAROS, the IKAROS-target genes are embedded in inactive chromatin conformation (not depicted) and they are characterized by the absence of PIC formation. To simplify the model, some of the proteins or complexes mentioned are not indicated.</p

IKAROS contributes to PP1α association with the NuRD-P-TEFb complex.

<p><b>A</b>) Protein co-immunoprecipitation of G1E2 nuclear extracts. Immunoprecipitations were carried out with IKAROS antibodies or isotype-matched immunoglobulins (IgG) and immunoblots were probed with PP1α or IKAROS antibodies; Input samples represent 2% of nuclear extracts; filled dot: non-specific band; <b>B, C, F</b>) ChIP assays were carried out with the antibodies labeled on the top of each panel; POL II: is an antibody against the N-terminal region of the large subunit of POL II and binds POL II in a phosphorylation-independent manner; PCTD: is an antibody against the CTD repeats phosphorylated at Ser2. Lin⁻ HPCs: bone marrow-derived lineage negative hematopoietic progenitor cells; Ik^WT lin⁻: <i>Ikaros</i> wild type lin⁻ HPCs; Ik^HT lin⁻: <i>Ikaros</i> heterozygote null lin⁻ HPCs; Ik^NULL lin⁻: <i>Ikaros</i> homozygote null lin⁻ HPCs; Nt-sh: non-target sh-RNA G1E2 clones; Ik-sh: <i>Ikaros</i>-specific sh-RNA (Ik-sh1 and Ik-sh2) G1E2 clones; DMSO: G1E2 cells treated for 30 min with 0.05% DMSO (diluent control); Calyculin A: G1E2 cells treated for 30 min with100 nM Calyculin A; <i>y</i>-axis: fold enrichments of <i>c-Kit</i> (−114 Kb; −0.5 Kb; TSS; +1 Kb, +5 Kb, +58 Kb ORF regions), <i>Alas2</i> (TSS) or <i>Gapdh</i> (TSS) regulatory regions relative to <i>Thp</i> promoter and input samples are plotted as the mean ± Standard Deviations (SD); a value of 1 (dotted lines) indicates no enrichment; n≥4; <i>Alas2</i>, <i>Gapdh</i> and <i>Thp</i> TSS regions were used as negative controls; <b>D, G</b>) Protein expression analysis. Western blot assays of total cell lysates were performed with non-target (Nt-sh) or <i>Ikaros</i>-specific (Ik-sh1 and Ik-sh2) sh-RNA G1E2 clones and with DMSO- or Calyculin A-treated G1E2 cells; the antibodies used are indicated on the right side of the panels; <b>E</b>) Gene expression profiles. RNA samples were retro-transcribed with random oligonucleotides to amplify nascent transcripts, which were used as templates for qPCR with intron-specific <i>c-Kit</i> (+49 Kb, +54 Kb, +67 Kb regions) or <i>Gapdh</i> (used as internal control) primer sets; <i>y</i> axis: relative nascent transcript enrichment levels; ratios are plotted as the mean ± SD of the measurements; n≥4. *: <i>P</i>≤0.05 by Student's <i>t</i>-test.</p

<i>c-Kit</i> POL II traveling ratio.

<p><i>c-Kit</i> traveling ratio values (as defined by the relative ratio of POL II density in gene ORF <i>vs.</i> promoter-proximal regions) were obtained by chromatin immunoprecipitation with POL II antibody, which recognizes the N-terminal region of the large subunit of POL II and binds POL II in a phosphorylation-independent manner and indicate the enrichment levels of <i>c-Kit</i> +5/−0.5 or +9,7/−0.5 regions relative to the control and the input samples (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004827#pgen-1004827-g002" target="_blank">Figure 2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004827#pgen-1004827-g003" target="_blank">3</a> legends); Ik^WT: <i>Ikaros</i> wild type HPCs; Ik^HT: <i>Ikaros</i> heterozygote null HPCs; Ik^NULL: <i>Ikaros</i> homozygote null lin⁻ HPCs; DMSO: Dimethyl sulfoxide-treated G1E2 cells (0.01% for 2 h); Fvp: Flavopiridol-treated G1E2 cells (100 nM for 2 h).</p><p><i>c-Kit</i> POL II traveling ratio.</p

The IKAROS-NuRD-P-TEFb complex assists POL II during transcription elongation.

<p>In panels A–C, G1E2 cells were treated for 2 h either with 0.01% DMSO (DMSO) or 100 nM Flavopiridol (Fvp). <b>A</b>) Protein expression analysis. Western blot assays of total cell lysates of DMSO- or Flavopiridol-treated G1E2 cells; ACTIN was used as an internal control; the antibodies used are indicated on the right side of the panels; <b>B, E</b>) Gene expression profiles. RNA samples were retro-transcribed with random oligonucleotides to amplify nascent transcripts, which were used as templates for qPCR with intron-specific <i>c-Kit</i> (+54 Kb, +67 Kb, +77 Kb, +79 Kb regions) or <i>Alas2</i> (+0.1 Kb, +21 Kb regions) primer sets; <i>Rnu2-1</i> (a Flavopiridol-insensitive gene) was used as internal control in panel B; <i>Gapdh</i> was used as internal control in panel E; <i>y</i> axis: relative nascent transcript enrichment levels; ratios are plotted as the mean ± Standard Deviation (SD) of the measurements; n≥4; <b>C</b>) ChIP assays were carried out with the antibodies indicated on the top of each panel; POL II: is an antibody against the N-terminal region of the large subunit of POL II and binds POL II in a phosphorylation-independent manner; PCTD: is an antibody against the CTD repeats phosphorylated at Ser2; <i>y</i>-axis: fold enrichments of <i>c-Kit</i> regions (−.5 Kb; TSS, +0.5 Kb, +1 Kb, +5 Kb, +9.7 Kb, +16.9 Kb ORF regions) relative to <i>Thp</i> promoter and input samples are plotted as the mean ± SD of the measurements; a value of 1 (dotted lines) indicates no enrichment; n≥4; <b>D</b>) Gene expression profiles. mRNA samples were retro-transcribed with oligo-dT nucleotides; cDNA was used as template for qPCR with gene-specific primer sets; <i>β-Actin</i> was used as internal control; <i>y</i> axis: relative mRNA enrichment levels; ratios are plotted as the mean ± SD of the measurements; n≥4. *: <i>P</i>≤0.05 by Student's <i>t</i>-test.</p

Immunoaffinity purification of Flag-HA-IKAROS complexes from Jurkat cells and their identification by LC-MS/MS analysis.

<p>The percentage values indicate the sequence coverage of the identified proteins; False Discovery Rate (FDR): 0%.</p><p>Immunoaffinity purification of Flag-HA-IKAROS complexes from Jurkat cells and their identification by LC-MS/MS analysis.</p

The simultaneous recruitment of P-TEFb and NuRD is IKAROS dose-dependent.

<p><b>A</b>) Protein co-immunoprecipitation of G1E2 total cell lysates. Immunoprecipitations were carried out with CDK9 antibodies; immunoblots were probed with Mi2 antibodies; Input samples represent 2% of protein extracts; <b>B, C</b>) Gene expression profiles. mRNA samples were retro-transcribed with oligo-dT nucleotides; cDNA was used as template for qPCR with gene-specific primer sets; β-<i>Actin</i> was used as internal control; <i>y</i> axis: relative mRNA transcript enrichment levels; ratios are plotted as the mean ± Standard Deviation (SD) of the measurements; n≥4; <b>D, E</b>) Chromatin Immunoprecipitation (ChIP). ChIP assays were carried out with the antibodies indicated on the top of each panel; POL II: is an antibody against the N-terminal region of the large subunit of POL II and binds POL II in a phosphorylation-independent manner; PCTD: is an antibody against the CTD repeats phosphorylated at Ser2; <i>y</i>-axis: fold enrichments of <i>c-Kit</i> (−114 Kb enhancer; −0.5 Kb; TSS; +1 Kb, +5 Kb, +9.7 Kb, +58 Kb ORF regions) or <i>Alas2</i> (TSS; used as negative control) regions relative to <i>Thp</i> promoter and input samples are plotted as the mean ± SD of the measurements; a value of 1 (dotted lines) indicates no enrichment. Lin⁻ HPCs: bone marrow-derived lineage negative hematopoietic progenitor cells; Ik^WT lin⁻: <i>Ikaros</i> wild type lin⁻ HPCs; Ik^HT lin⁻: <i>Ikaros</i> heterozygote null lin⁻ HPCs; Ik^NULL lin⁻: <i>Ikaros</i> homozygote null lin⁻ HPCs; Nt-sh: non-target sh-RNA G1E2 clones; Ik-sh: <i>Ikaros</i>-specific sh-RNA (Ik-sh1 and Ik-sh2) G1E2 clones. *: <i>P</i>≤0.05 by Student's <i>t</i>-test; <b>F, G</b>) Gene expression profiles. RNA samples were retro-transcribed with random oligonucleotides to amplify nascent transcripts, which were used as templates for qPCR with intron-specific <i>c-Kit</i> (+0.5 Kb and +79 Kb regions), <i>Alas2</i> (+0.1 Kb and +21 Kb regions) or <i>Gapdh</i> (used as internal control) primer sets; <i>y</i> axis: relative nascent transcript enrichment levels; ratios are plotted as the mean ± Standard Deviation (SD) of the measurements; n≥4; <b>H</b>) Sequential ChIP (re-ChIP) assays carried out on G1E2 cells. Mi2, CDK9 or IKAROS antibodies were used for the first ChIP; IKAROS, CDK9, Mi2 antibodies or IgG controls were used for the second ChIP; left panel: representative examples of semi-quantitative PCR of re-ChIP sample carried out with primer sets specific for <i>c-Kit</i> TSS or <i>Thp</i> promoter; right panel: quantitative re-ChIP analysis carried out with the percent input method; re-ChIP samples were used as templates for quantitative PCR with primer sets specific for <i>c-Kit</i> TSS or <i>Thp</i> promoter; <i>y</i>-axis: fold enrichments of <i>c-Kit</i> TSS or <i>Thp</i> promoter (used as negative control) regions in re-ChIP samples relative to input samples are plotted as the mean ± SD of the measurements; n = 3.</p

IKAROS interacts with the newly characterized NuRD-P-TEFb complex.

<p><b>A</b>) Purification of Flag-HA-Ik associated proteins. Nuclear extracts from Jurkat cells carrying the pOZ-N-Flag-HA-IKAROS-IRES-IL2R vector and expressing a double tagged IKAROS (Flag-HA-Ik) or Jurkat cells expressing the pOZ-N-Flag-HA-IRES-IL2R empty vector (Mock) were used for sequential immunoaffinity purification using Flag- followed by HA-conjugated matrix. A fraction of the purified complexes were loaded on SDS-PAGE and silver stained; E1: first HA elution; E2: second HA elution; MW: molecular weights (in KDa); the Flag-HA-Ik protein is indicated by an arrow; <b>B, C</b>) Molecular weight fractionation of the IKAROS-associated complexes. Flag-HA-Ik immunoaffinity purified complexes (panel B) or nuclear extracts (panel C) were fractionated using a Superose 6 10/300GL column; 0.5 ml fractions were collected, TCA-precipitated and loaded on SDS-PAGE for western blot analysis; immunoblots were probed with the antibodies indicated at the bottom or on the right side of the panels; the 37 KDa APE1 nuclear protein was used as control; <b>D</b>) Protein co-immunoprecipitation of Jurkat cell nuclear extracts. Immunoprecipitations were carried out with the antibodies indicated on top of each panel; these antibodies were specific for: IKAROS, the general P-TEFb activator BRD4, NuRD-associated proteins (Mi2, RBBP4, MTA2, MBD3) or P-TEFb components (CDK9, CYCT1). Immunoblots were probed with the antibodies indicated at the bottom of the panels. Input samples represent 2% of nuclear extracts; IgG: isotype-matched immunoglobulins; asterisk: IgG light chains; filled dots: non-specific bands; n≥3; <b>E</b>) The nuclear protein PCNA was used as negative control for the immunoprecipitation procedure; <b>F</b>) Summary of the most relevant protein-protein interactions; +: strong interaction; +/−: weak interaction; −: no interaction; nd: not determined.</p