Members of the NuRD chromatin remodeling complex interact with AUF1 in developing cortical neurons

Abstract

Chromatin remodeling plays an important role in coordinating gene expression during cortical development, however the identity of molecular complexes present in differentiating cortical neurons that mediate the process remains poorly understood. The A + U-rich element-binding factor 1 (AUF1) is a known regulator of messenger RNA stability and also acts as a transcription factor upon binding to AT-rich DNA elements. Here we show that AUF1 is specifically expressed in subsets of proliferating neural precursors and differentiating postmitotic neurons of the developing cerebral cortex. Moreover, AUF1 is coexpressed with histone deacetylase 1 (HDAC1) and metastasis-associated protein 2 (MTA2), members of the nucleosome remodeling and histone deacetylase complex. AUF1 specifically and simultaneously binds to HDAC1, MTA2, and AT-rich DNA element, its gene regulatory function is modulated by the extent of histone acetylation and in animals lacking AUF1, the composition of the complex is modified. These results suggest that AUF1 is involved in integrating genetic and epigenetic signals during cortical development through recruiting HDAC1 and MTA2 to AT-rich DNA elements.

Figure 1.

Expression of AUF1 proteins in the developing and adult rat brain. Representative Western blot analysis showing the relative abundance of the various isoforms at the selected ages. Star indicates the nonspecific band recognized by the anti-AUF1 antibody.

Figure 2.

The proliferative state of AUF1-expressing cells in the rat brain. The bivariate dot density plot derived from FACS analysis shows the expression(s) of PCNA and AUF1 in the mostly proliferating neural precursor/progenitor population isolated from the E13 rat telencephalon (A) and their predominantly postmitotic/differentiating counterparts isolated from E19 rat cortex (B). Cell population in the upper right quadrant represents the contribution of proliferating AUF1+ cells to the total cell population at the 2 developmental ages. The developmental changes in the percentage distribution among the various cell populations are tabulated (C). Upper-left (UL) represents AUF1⁺/PCNA⁻, upper-right (UR) represents AUF1⁺/PCNA⁺, lower-left (LL) represents AUF1⁻/PCNA⁻, and lower-right (LR) represents AUF1⁻/PCNA⁺ fraction, respectively.

Figure 3.

The developmental phenotype of AUF1-expressing cells in the rat brain. Double immunohistochemistry (A–C) of E14 rat coronal sections show the location of proliferating Phospho-Histone H3+/AUF1+ cells. At this age, most nestin+ cells that are located in the VZ and SVZ express AUF1 (D–F). At E18, most AUF1+ cells that are located in the MZ and CP of the developing cortex express TuJ1 (G–I). After birth, the number of AUF1+ cells decreases substantially and the remaining AUF1+ cells express MAP2 (J–L) at P2. LV = lateral ventricle; IMZ = intermediate zone. Scale bars in (A–F) = 200 μm and in (G–L) = 100 μm.

Figure 4.

Pattern of coexpression of AUF1, HDAC1, and MTA2 in the developing rat brain. Anatomical maps of coronal sections of the developing rat brain at ages E14, E18, and P2 (A) summarizing the patterns of coexpression (yellow colored areas). Left hemispheres: AUF1/HDAC1; right hemispheres: AUF1/MTA2. Abbreviations: Bta = Basal telencephalon; Spt = septal neuroepithelium; Lv = lateral ventricle; Str = striatum; Cx = cortex. Representative double-immunohistochemical images showing the pattern of AUF1, HDAC1, and MTA2 expression in the developing rat brain (B). Coronal sections from E14, E18 and P2 rat brains were immunostained using a combination of AUF1 and HDAC1 (upper row) and AUF1 and MTA2 (lower row) antibodies. Abbreviations: Cx, cortex; LV, lateral ventricle; IMZ, intermediate zone. Scale bar = 1 mm. Graphs showing the extent of coexpression (AUF1/HDAC1 and AUF1/MTA2) from E14 to P2 in the developing rat cortex. The bar graphs show the ratio of red (AUF1), green (HDAC1 or MTA2), and yellow (double-positive) areas expressed as percentage of the total area scanned (C). Vertical axis = percentage of total color pixels. N = 4.

Figure 5.

In vitro and in vivo interactions of AUF1 with HDAC1, MTA2, and AT-rich DNA. Nuclear extracts (NE) were prepared from the E18 rat cortex and immunoprecipitated with either AUF1 or control antibody. Following immunoprecipitation, bound (B) and free (F) fractions were analyzed by Western blots using specific antibodies. The identities of AUF1-, MTA2-, and HDAC1-immunoreactive bands are marked on the left. Molecular weight (MW) is shown in kDa on the right side. Star indicates a nonspecific band (A). The DAPSTER assay was performed using nuclear extracts prepared from the E18 rat cortex. DNA affinity assay was either carried out in the absence of competitors (-), or in the presence of the specific competitor rAT or control rAT_MUT dsDNA. The presence of the AUF1, HDAC1 and MTA2 in the DNA–protein complex was determined by Western blot using specific antibodies. The identities of AUF1-, MTA2- and HDAC1-immunoreactive bands are marked on the left (B). A ∼150-bp DNA fragment containing the rAT DNA region was amplified from samples that were immunoprecipitated with AUF1, HDAC1 or MTA2 antibodies and PCR amplicons were separated on agarose gels. No DNA was amplified from samples that were immunoprecipitated using the preimmune serum to AUF1 or a control antibody (EGFR). The ChIP assay was performed on postnatal (P2) rat cortical tissue. Precipitated DNA fragments were amplified by PCR, then the product of the nested (2nd) PCR was subjected to agarose gel electrophoresis and DNA fragments were visualized by ethydium bromide staining under UV light. Input sample indicates the nonimmunoprecipitated positive control; plasmid DNA containing the rAT region used as template for PCR amplification was used as another positive control (+). For the negative control (-) we used the EGFR-precipitated sample (C).

Figure 6.

The effect of TSA treatment on AUF1-dependent reporter gene activity. MEF-AUF1^+/+ and MEF-AUF1^−/− cells were transfected with reporter plasmids pTK-rAT-Ren or pTK-rAT_MUT-Ren and treated with TSA (gray columns) or with vehicle (black columns). The results were normalized by subtracting values measured in cultures transfected with pTK-rAT_MUT-Ren from values obtained from cultures transfected with pTK-rAT_MUT-Ren and expressed as RLU. The lack of AUF1 in MEF-AUF1^−/− cells resulted in a significant loss of the AT-rich DNA dependent repressor of AUF1 and a loss of the effect of TSA treatment. Star = P ≤ 0.05; N = 4, ±SD.

Figure 7.

AUF1 proteins interact with both the mAT^ENK region and the NuRD complex and control chromatin remodeling. Semiquantitative ChIP assay was performed using whole brain tissue from P2 wild-type (WT) or AUF1 knockout (KO) mouse brains. A 274-bp DNA fragment containing the AT-rich regulatory region of the mouse ENK gene, or a negative control region was amplified from samples that were immunoprecipitated with anti-AUF1, anti-HDAC1, anti-SATB2 (Szemes et al. 2006), or anti-MTA2 antibodies, or normal rabbit serum as a negative control (not shown). As a negative control for AUF1-binding, we used a region in the GAPDH gene (control shown). After amplification by PCR, the product was subjected to agarose gel electrophoresis and DNA fragments were visualized by SYBR Safe staining (A). Images were collected using the Fuji 3000 LAS intelligent dark box equipped with a CCD camera, and band densities were quantified with the Image Gauge software. Raw values were normalized to the input and background, and then the fold change difference between KO to WT was calculated. Star = P ≤ 0.05; N = 4. The average values from 3 independent experiments are shown with error bars indicating ± SD (B).