QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Borghi, S. Articles by Ferrari, S. Search for Related Content PubMed PubMed Citation Articles by Borghi, S. Articles by Ferrari, S.

The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4

Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica, Università di Modena e Reggio Emilia, Via G. Campi 287, 41100 Modena, Italy

View larger version (70K): [in a new window]   Fig. 1. Cellular localization of GFP/MEF2 fusion proteins. (A) Cellular localization of GFP (a), GFP/MEF2A (b) and GFP/MEF2C (c) in transfected C2.7 myoblasts; Hoechst staining of nuclei is shown in d-f: the arrows indicate the nuclei corresponding to green fluorescent cells. (B) Cellular localization of GFP (a), GFP/MEF2A-471 (b), GFP/MEF2C-429 (c), GFP/MEF2C-411 (d), GFP/MEF2C-399 (e) in transfected C2.7 myoblasts; Hoechst staining of nuclei is shown in panels f-j: the arrows indicate the nuclei corresponding to green fluorescent cells. Bar, 5 µm. (C) Linear representation of the GFP/MEF2 constructs used. The relative distribution of GFP fluorescence in the nuclei (N), in the cytoplasm (C), or in the nuclei and cytoplasm (N&C) of transfected C2.7 cells is shown for each construct. NLS refers to the C-terminal sequence encompassing aa 472-507 in MEF2A, or aa 430-466 in MEF2C, where the bipartite nuclear localization signal (NLS) is located.

View larger version (51K): [in a new window]   Fig. 2. Cellular localization of GFP fused to selected C-terminal domains of MEF2A and MEF2C. (A) Cellular localization of GFP (a), GFP/MEF2A472-507 (b), GFP/MEF2C430-466 (c), GFP/MEF2C401-466 (d), GFP/MEF2C389-411 (e), GFP/MEF2C401-429 (f) in transfected C2.7 myoblasts. Bar, 5 µm. (B) Nuclear/cytoplasmic ratio of GFP fluorescence corresponding to the GFP/MEF2 constructs shown in A. (C) Linear representation of the GFP/MEF2 constructs used. NLS refers to the C-terminal sequence encompassing aa 472-507 in MEF2A or aa 430-466 in MEF2C, where the bipartite nuclear localization signal (NLS) is located. (D) Sequence alignment of the C-terminal region of human MEF2A and MEF2C. Amino acids corresponding to the bipartite nuclear localization signal are shown in bold. The sequence of MEF2C containing a putative nuclear retention signal is underlined.

View larger version (46K): [in a new window]   Fig. 3. Cellular localization of HDAC4 in C2.7 cells co-transfected with MEF2 proteins. (A) Cellular localization of Myc-tagged HDAC4 and HA-tagged HDAC5 in proliferating C2.7 myoblasts (a and b, respectively) as well as in differentiated C2.7 myotubes (c and d, respectively); corresponding Hoechst stained nuclei are indicated by arrows in e-h. (B) Cellular localization of Myc-tagged HDAC4 (red) in C2.7 myoblasts co-transfected with pGFP/MEF2A (a), pGFP/MEF2C (b), pGFP/MEF2A-471 (c), pGFP/MEF2C-429 (d) and pGFP/MEF2C-399 (e). Green fluorescence of the same cells co-expressing the indicated GFP/MEF2 fusion proteins is shown in f-j. Bar, 4 µm.

View larger version (71K): [in a new window]   Fig. 4. Cellular localization of HDAC4 in cells co-transfected with NF-YB, GFP or MEF2NLS. Myc-tagged HDAC4 (red) localization in C2.7 myoblasts co-transfected with NF-YB (a,d), pEGFP-C3 (b,e) and pGFP/MEF2A472-507 (c,f). Bar, 4 µm.

View larger version (31K): [in a new window]   Fig. 5. Effect of MEF2 proteins on nuclear/cytoplasmic distribution of HDAC4. Myc-tagged HDAC4 (HDAC4) subcellular distribution was analyzed in C2.7 myoblasts transfected with pcHDAC4-Myc alone, or in combination with pGFP/MEF2A (MEF2), pGFP/MEF2A-471 (MEF2NLS), pGFP/MEF2A472-507 (MEF2NLS).

View larger version (20K): [in a new window]   Fig. 6. Western blot analysis of HDAC4 localization. Nuclear protein extracts (lanes 1-3) and cytoplasmic protein extracts (lanes 4-6) from C2.7 myoblasts transfected with pcHDAC4-Myc and with pFLAG/MEF2C (lanes 1,4) or with pFLAG/MEF2CNLS (lanes 2,5) were subjected to western blot analysis with anti-Myc antibody. Nuclear protein extracts from cells transfected with pcHDAC4-Myc, alone (lane 7) or in combination with either pFLAG/MEF2C (lane 8) or pFLAG/MEF2CNLS (lane 9), were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were subjected to western blot analysis with anti-Myc antibody. Lanes 3, 6 and 10 (C), are control lanes showing protein extracts from mock transfected cells.