Isolation of human NURF: a regulator of Engrailed gene expression

Abstract

The modification of chromatin structure is an important regulatory mechanism for developmental gene expression. Differential expression of the mammalian ISWI genes, SNF2H and SNF2L, has suggested that they possess distinct developmental roles. Here we describe the purification and characterization of the first human SNF2L-containing complex. The subunit composition suggests that it represents the human ortholog of the Drosophila nucleosome-remodeling factor (NURF) complex. Human NURF (hNURF) is enriched in brain, and we demonstrate that it regulates human Engrailed, a homeodomain protein that regulates neuronal development in the mid-hindbrain. Furthermore, we show that hNURF potentiates neurite outgrowth in cell culture. Taken together, our data suggess a role for an ISWI complex in neuronal growth.

Fig. 1. Biochemical isolation of the hNURF SNF2L-associated complex. (A) Purification scheme. fSNF2L-HEK293 nuclear extract was fractionated by chromatography as described in Materials and methods. The horizontal and diagonal lines indicate stepwise and gradient elution, respectively. (B) Fractions immunoprecipitated from Superose 6 elutions using M2 anti-Flag beads were resolved on an SDS-polyacrylamide (4–12%) gel, and proteins were visualized by silver staining. Molecular masses of marker proteins (left) and polypeptide masses of associated subunits (right) are indicated.

Fig. 2. Composition of the novel SNF2L-associated complex. (A) Schematic of the SNF2L complex subunits. (B) Specificity of BPTF-N antibody for cDNAs expressing Fac1 (BPTF 1–2622) and full-length BPTF. (C) Immunoblot analysis of fractions from M2 anti-Flag beads resolved on an SDS–polyacrylamide (4–12%) gel (I, input; FT, flow through; W, wash; B, bound). Antibodies used for immunoblot are indicated (left). (D) Endogenous hNURF immunoprecipitated from HEK293 partially purified nuclear extract. Immunoblot analysis of immunoprecipitation was resolved on an SDS–polyacrylamide (4–12%) gel. Antibodies used for immunoprecipitation (IP) are indicated across the top, while antibodies used for immunoblot (IB) are on the left. Input represents 5% of IP. Flow through (FT) and bound (B) fractions are as indicated. (E) hNURF molecular mass is ∼1 MDa. Immunoblot of elutions from the Superose 6 gel filtration column from partially purified HEK293 nuclear extract. Fractions (fxn) as well as void (Vo) and thyroglobulin (670 kDa) molecular weight markers are indicated (top).

Fig. 3. hNURF purified with a one-step purification protocol. Mock and Flag-BPTF eluted fractions were fractionated by 8–16% SDS–PAGE followed by silver staining and western analysis. BPTF, SNF2L, RbAP48 and RbAP46 poplypeptides are indicated on the right. Western blot analysis performed in parallel confirms the identification of the polypeptides. Polypeptides marked with asterisks are contaminants also present in the mock elution. Molecular weight markers are indicated on the left.

Fig. 4. hNURF is a chromatin remodeler. (A) hNURF remodels chromatin. Autoradiograph of the restriction endonuclease-coupled remodeling assay demonstrating chromatin-remodeling activity. Mock IP failed to demonstrate any decrease in the uncut (U) array in an ATP-dependent manner (compare top and bottom panel of lane 1). Addition of purified hNURF results in a qualitative decrease in the uncut (U) array compared with mock (compare lanes 1 and 2, top panel). This activity is ATP dependent (compare lane 2 top and bottom panels). (B) hNURF addition results in an ∼2.5-fold decrease in the fraction of the uncut array. Densitometric analysis of restriction endonuclease-coupled remodeling. The graph represents the averages of the quantified fraction of the uncut array (U/U + C) from three experiments. Standard errors for the experiments are as indicated in the graph. (C) hNURF exhibits dose-dependent chromatin-remodeling activity. Graphical representation of densitometric analysis of assays over 0, 1.25, 2.5, 5 and 50 fmol of hNURF with or without ATP. Points represent the average of three reactions, with standard error as indicated. (D) Time course of hNURF-mediated chromatin remodeling. Restriction enzyme accessibility for buffer + ATP, 50 fmol hNURF-ATP or 50 fmol hNURF + ATP. Points represent the average of three reactions, with standard error as indicated.

Fig. 5. hNURF exhibits intrinsic nucleosome-dependent ATPase activity. (A) hNURF exhibits DNA- and nucleosome-stimulated ATPase activity. ATPase activity assays performed on [γ-³²P]ATP followed by separation of free phosphate (Pi) from ATP by PEI-cellulose TLC and autoradiography of TLC plates. hNURF activity is stimulated by supplementing the reaction with DNA (50 ng) and is potentiated further by nucleosomes (50 ng); compare lanes 4, 5 and 6. (B) hNURF activity responds to increasing amounts of nucleosomes. A 25 fmol concentration of hNURF or buffer was incubated with increasing amounts (0, 50, 100 or 250 ng) of either free DNA or free nucleosomes. Points represent the average of three reactions, with standard error as indicated. (C) hNURF exhibits a dose-dependent ATPase activity in the presence of nucleosomes. ATPase activity of three different hNURF concentrations (20, 50 and 100 fmol) with buffer, free DNA or nucleosomal DNA. Points represent the average of three reactions, with standard error as indicated. (D) Time course of hNURF ATPase activity. hNURF (100 fmol) was incubated with buffer, free DNA or nucleosomal DNA, and reactions were assayed for ATPase activity at the indicted times. Points represent the average of three reactions, with standard error as indicated.

Fig. 6. SNF2L and BPTF show overlapping expression in adult mouse brain. Adjacent sections from an adult mouse brain were hybridized with SNF2L (A, E and G) and BPTF (B, F and H) antisense RNA probes. Both SNF2L and BPTF were highly expressed throughout all regions of the hippocampus (CA1–4) and in the granule cell layer of the cerebellum (A and B dark field). The specificity of SNF2L and BPTF antisense probes is demonstrated by the lack of hybridization of corresponding sense probes (C and D). Higher magnifications of sections shown in (A) and (B) counterstained with cresyl violet demonstrate the expression of SNF2L and BPTF in the granule and Purkinje cell layer of the cerebellum (E and F) and in the pyramidal neurons of the hippocampus (G and H). Scale bars, 2 mm in (B); 50 µm in (F) and (H); gc, granule cells; p, Purkinje neurons; CA1 pyr, CA1 pyramidal neurons.

Fig. 7. hNURF localizes to and regulates human Engrailed. (A) ChIP assays localize hNURF specifically to engrailed-1 (en-1) and engrailed-2 (en-2) promoters. Chromatin from HEK293 cells was subjected to formaldehyde cross-linking followed by sonication and immunoprecipitation with non- specific, BPTF and SNF2L antibodies. After extensive washes, bound DNA was eluted and subjected to PCR with the indicated promoter primers. A specific and reproducible signal at the engrailed promoters comparable with input (In) was detected in the BPTF and SNF2L IPs but not in the non-specific IgG. Input fractions represent 1% of the total IP. Multiple primer pairs from each promoter were employed. Non-specific promoters include presenilin-1, znf261, UXT, APP and MECP2 as indicated. A representative set is displayed. (B) siRNA-mediated depletion of SNF2L. HEK293 cells transfected with either mock, SNF2L-specific siRNAs or non-specific siRNAs. Extract from cells 72 h after transfection were prepared and separated by SDS–PAGE followed by western analysis for SNF2L levels. The SNF2L-specific siRNA depletes SNF2L to undetectable levels compared with mock and non-specific siRNAs (compare lane 2 with lanes 1 and 3, top panel). Lower panel: loading control for protein levels determined by RbAP48 levels. (C) Depletion of SNF2L leads to a significant decrease in en-1 and en-2 transcripts. Total RNA was extracted from SNF2L-depleted and control HEK293 cells and subjected to reverse transcription followed by real-time PCR. Levels of transcripts were quantified and normalized to β-actin levels using the ABI7000 sequence detection system. Genes lacking hNURF at their promoters as determined by ChIP were used as controls. The bar graph depicts the average of three independent experiments. Standard errors are indicated. (D) Depletion of BPTF leads to a significant decrease in en-1 transcript. Experiments were performed as in (C). The bar graph depicts the averages of three independent experiments. Standard errors are indicated.

Fig. 8. SNF2L promotes neurite outgrowth. N1E-115 cells were co-transfected transiently with vector (A and D), f-SNF2L-WT (B and E) or f-SNF2L-K213R (C and F) along with a β-galactosidase expression plasmids in a 10:1 ratio. After transfection, cells were cultured for 30 h in normal (non-inducing) growth medium and then fixed for immunofluorescence staining (Flag = green, β-galactosidase = red). Differentiated cells were identified as cells in which the length of the neurite extensions was at least twice the diameter of the cell body. The fold increase in neurite outgrowth in f-SNF2L-transfected cells compared with vector-transfected cells was calculated from cell counts (600 cells) obtained from three different experiments (G) and is statistically significant at P < 0.005. Scale bars, 50 µm.