The Disordered Amino-Terminus of SIMPL Interacts with Members of The 70-kDa Heat-Shock Protein Family

The p65 coactivator SIMPL is a small protein that lacks any conserved domains of known function. To better understand regulation of SIMPL activity, we sought to identify novel SIMPL interacting proteins using mass spectrometry analysis of SIMPL containing complexes. Two members of the 70-kDa heat-shock protein family, Hsp70 and Hsc70, were identified as SIMPL binding proteins. Subsequent immunocomplexing assays confirmed this interaction and demonstrated that the amino-terminus of SIMPL is required for this interaction. Using a combination of amino acid composition analysis, PONDR® VL-XT prediction, charge-hydropathy plots, and cumulative distribution functions, the amino-terminal region of both mouse and human SIMPL proteins was predicted to be intrinsically disordered. These data, taken together, suggest that Hsp70/Hsc70 bind the intrinsically disordered amino-terminal region of SIMPL to stabilize the protein and thereby regulate its activity. Understanding the regulation of SIMPL through its interaction with Hsp70/Hsc70 may serve as a novel means of modulating tumor necrosis factor alpha signaling

Use of Genome Engineering to Create Patient Specific MLL Translocations in Primary Human Hematopoietic Stem and Progenitor Cells.

One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the MLL gene and two different known translocation partners (AF4 and AF9), which resulted in specific chromosomal translocations in K562 cells as well as primary hematopoietic stem and progenitor cells (HSPCs). The initiation of a specific MLL translocation in a small number of HSPCs likely mimics the leukemia-initiating event that occurs in patients. In our studies, the creation of specific MLL translocations in CD34+ cells was not sufficient to transform cells in vitro. Rather, a variety of fates was observed for translocation positive cells including cell loss over time, a transient proliferative advantage followed by loss of the clone, or a persistent proliferative advantage. These studies highlight the application of genome engineering tools in primary human HSPCs to induce and prospectively study the consequences of initiating translocation events in leukemia pathogenesis

Tumor Necrosis Factor Alpha Induction of NF-κB Requires the Novel Coactivator SIMPL

A myriad of stimuli including proinflammatory cytokines, viruses, and chemical and mechanical insults activate a kinase complex composed of IκB kinase β (IKK-β), IKK-α, and IKK-γ/N, leading to changes in NF-κB-dependent gene expression. However, it is not clear how the NF-κB response is tailored to specific cellular insults. Signaling molecule that interacts with mouse pelle-like kinase (SIMPL) is a signaling component required for tumor necrosis factor alpha (TNF-α)-dependent but not interleukin-1-dependent NF-κB activation. Herein we demonstrate that nuclear localization of SIMPL is required for type I TNF receptor-induced NF-κB activity. SIMPL interacts with nuclear p65 in a TNF-α-dependent manner to promote endogenous NF-κB-dependent gene expression. The interaction between SIMPL and p65 enhances p65 transactivation activity. These data support a model in which TNF-α activation of NF-κB dependent-gene expression requires nuclear relocalization of p65 as well as nuclear relocalization of SIMPL, generating a TNF-α-specific induction of gene expression

SIMPL enhancement of tumor necrosis factor-α dependent p65-MED1 complex formation is required for mammalian hematopoietic stem and progenitor cell function.

Significant insight into the signaling pathways leading to activation of the Rel transcription factor family, collectively termed NF-κB, has been gained. Less well understood is how subsets of NF-κB-dependent genes are regulated in a signal specific manner. The SIMPL protein (signaling molecule that interacts with mouse pelle-like kinase) is required for full Tumor Necrosis Factor-α (TNFα) induced NF-κB activity. We show that SIMPL is required for steady-state hematopoiesis and the expression of a subset of TNFα induced genes whose products regulate hematopoietic cell activity. To gain insight into the mechanism through which SIMPL modulates gene expression we focused on the Tnf gene, an immune response regulator required for steady-state hematopoiesis. In response to TNFα SIMPL localizes to the Tnf gene promoter where it modulates the initiation of Tnf gene transcription. SIMPL binding partners identified by mass spectrometry include proteins involved in transcription and the interaction between SIMPL and MED1 was characterized in more detail. In response to TNFα, SIMPL is found in p65-MED1 complexes where SIMPL enhances p65/MED1/SIMPL complex formation. Together our results indicate that SIMPL functions as a TNFα-dependent p65 co-activator by facilitating the recruitment of MED1 to p65 containing transcriptional complexes to control the expression of a subset of TNFα-induced genes

<i>MLL</i>, <i>AF4</i>, and <i>AF9</i> TALEN design.

<p>(A, C) TALEN pairs (bold sequence) were designed to target known translocation break points (underlined) within the <i>MLL</i>, <i>AF4</i>, and <i>AF9</i> genes. (B, D) Following nucleofection into K562 cells, cutting by each TALEN pair was measured using the surveyor assay, which detects indels within the target region that result from imprecise repair of a DNA double strand break. Asterisks denote cleaved PCR products.</p

Co-expression of <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs in primary human CD34+ cells induces <i>MLL</i> translocations.

<p>(A, B) Primary human CD34+ cells isolated from umbilical cord blood were nucleofected with <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs. Genomic DNA was isolated at the indicated time point and used for PCR analysis for <i>MLL</i> translocations. (C, D) PCR products were isolated and sequenced to confirm translocation products. Nucleotide sequences shown are a composite alignment of PCR products from multiple experiments showing a variety of distinct translocations. Underlines denote TALEN binding sites.</p

CD34+ cells nucleofected with <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs display enhanced replating potential in semi-solid medium.

<p>Colony forming assays were performed to assess the effect of <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs on the replating efficiency of CD34+ cells in semi-solid medium. The bars in Panel A represent the mean number of colonies generated per 10⁴ seeded cells. Panel B shows the morphology of secondary colonies generated by CD34+ cells nucleofected with <i>MLL</i> and <i>AF4</i> or <i>AF9</i> TALENs: diffuse colony representing ~80% (left) and compact colony representing ~20% (right) of all colonies.</p

Survival advantage for CD34+ cells containing an <i>MLL-AF4</i> translocation.

<p>Primary human CD34+ cells isolated from umbilical cord blood were nucleofected with <i>MLL</i> and <i>AF4</i> TALENs. The population was immediately divided into 20 subcultures (50,000 cells per well), which were maintained over time. Genomic DNA was isolated at the indicated time points and used for semi-quantitative PCR analysis for <i>MLL</i> translocations (A) and qPCR for the MLL-AF4 translocation (B). (-- indicates subcultures nucleofected with GFP as negative control)</p

FISH analysis shows <i>MLL</i> translocations in human CD34+ cells induced by <i>MLL</i> and <i>AF4</i> TALENs.

<p>(A-D) FISH analysis using an <i>MLL</i> break apart probe was performed on cells from subcultures 9 (A, B) and 20 (C) as well as a negative control culture (D) to evaluate the presence of <i>MLL</i> chromosomal translocations. Two hundred cells were analyzed for each sample. Representative images are shown. Red arrows indicate cells positive for an <i>MLL</i> translocation. Panel B shows a cell from subculture 9 in metaphase that was noted to have an <i>MLL</i> translocation.</p