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. 2014 Dec 16;111(50):17809-14.
doi: 10.1073/pnas.1414004111. Epub 2014 Dec 1.

Self-assembled FUS binds active chromatin and regulates gene transcription

Liuqing Yang  1 , Jozsef Gal  1 , Jing Chen  1 , Haining Zhu  2
Affiliations

Self-assembled FUS binds active chromatin and regulates gene transcription

Liuqing Yang et al. Proc Natl Acad Sci U S A. .

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Fused in sarcoma (FUS) is a DNA/RNA binding protein and mutations in FUS cause a subset of familial ALS. Most ALS mutations are clustered in the C-terminal nuclear localization sequence of FUS and consequently lead to the accumulation of protein inclusions in the cytoplasm. It remains debatable whether loss of FUS normal function in the nucleus or gain of toxic function in the cytoplasm plays a more critical role in the ALS etiology. Moreover, the physiological function of FUS in the nucleus remains to be fully understood. In this study, we found that a significant portion of nuclear FUS was bound to active chromatin and that the ALS mutations dramatically decreased FUS chromatin binding ability. Functionally, the chromatin binding is required for FUS transcription activation, but not for alternative splicing regulation. The N-terminal QGSY (glutamine-glycine-serine-tyrosine)-rich region (amino acids 1-164) mediates FUS self-assembly in the nucleus of mammalian cells and the self-assembly is essential for its chromatin binding and transcription activation. In addition, RNA binding is also required for FUS self-assembly and chromatin binding. Together, our results suggest a functional assembly of FUS in the nucleus under physiological conditions, which is different from the cytoplasmic inclusions. The ALS mutations can cause loss of function in the nucleus by disrupting this assembly and chromatin binding.

Keywords: amyotrophic lateral sclerosis; chromatin binding; fused in sarcoma; self-assembly; transcription.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
ALS mutations reduce FUS binding with active chromatin. (A) The soluble (S) and chromatin-bound (CB) fractions were subjected to SDS/PAGE and Western blot with FUS and histone H3 antibodies. (B) HEK cell lysates were separated into cytoplasmic (C), membrane (M), nuclear soluble (NS), chromatin-bound (CB), and cytoskeletal (Sk) fractions using a Pierce Subcellular Protein Fractionation kit. Each fraction was subjected to SDS/PAGE and Western blot with indicated antibodies. Quantification of FUS in each fraction out of the total FUS amount is shown as percentage values. (C) The distribution of GST-tagged wild-type FUS and ALS mutants R521G and R495X was examined by the fractionation method as in B. Percentage values represent the relative abundance of FUS in each fraction out of the total FUS amount. The ratio of CB and NS was quantified and the results from three independent experiments are presented. *P < 0.02. (D) Active and inactive chromatin domains were separated as shown in the flowchart in Fig. S1C. All fractions (S1, W1, S2, W2, E1, E2, and P) were subjected to SDS/PAGE and Western blot with indicated antibodies (Upper) or agarose gel electrophoresis and ethidium bromide staining (Lower). (E) The association of GFP-tagged wild-type FUS and ALS mutants R521G and R495X with active chromatin was examined using the salt elution protocol as in D. The ratio of FUS in E1 and S2 was quantified and the results from three independent experiments are presented. *P < 0.05. The antibodies used in Western blot were: copper-zinc superoxide dismutase (SOD1), a primarily cytoplasmic protein; transcription factor Sp1, a nuclear soluble protein; histone H3, a chromatin-bound protein; and histone H1, a protein associated with inactive chromatin.
Fig. 2.
Fig. 2.
The QGSY-rich region is required for FUS chromatin binding. (A) The full-length (FL) and the truncated FUS proteins were subjected to the chromatin-bound protein isolation protocol by SDS and benzonase extraction as shown in Fig. S1A. The chromatin-bound and soluble fractions were subjected to SDS/PAGE and Western blot with indicated antibodies. (B) The intranuclear distribution of the EGFP-tagged full-length FUS and FUS 165–526 lacking the QGSY-rich region was examined by confocal microscopy. Green, GFP-tagged FUS; blue, DAPI staining of DNA; and arrows, nucleoli.
Fig. 3.
Fig. 3.
The QGSY-rich region is required for FUS transcription activation but not splicing regulation. (A) The transcription activation by FUS was monitored by a dual luciferase reporter assay. The ratio of Firefly and Renilla luciferase activities in the presence of full-length FUS or FUS 165–526 lacking the QGSY-rich region. The results from three independent experiments are presented. NT, only reporter plasmid transfected. *P < 0.01. (B) The transcription activation of endogenous SMYD3 gene by FUS as measured by real-time PCR. The results from three independent experiments are presented. *P < 0.05. (C) The mRNA splicing regulation by FUS was monitored by minigene splicing assay. (Left) The diagram of alternative splicing of the E1A and insulin receptor minigenes. Dash lines indicate exon inclusion, whereas solid lines indicate exon exclusion in splicing products. (Middle) Images of the ethidium bromide-stained gels show minigene transcript variants in HEK cells expressing full-length FUS or FUS 165–526 lacking the QGSY-rich region. The major exon inclusion and exon exclusion transcripts are indicated, respectively. (Right) The ratio of exon inclusion and exon exclusion transcripts was quantified and results from three independent experiments are presented in the bar graph. *P < 0.01. N.S., no significant difference between full-length FUS and FUS 165–526.
Fig. 4.
Fig. 4.
The QGSY-rich region mediates FUS self-assembly. (A) Diagram of FUS 165–526 tagged by tetrameric DsRed2 or monomeric DsRed–Monomer. (B) Tetrameric DsRed2 restored the binding of FUS 165–526 to chromatin, whereas monomeric DsRed-tagged FUS 165–526 was not detected in the chromatin-bound fraction. HEK cells were transfected with DsRed2–FUS 165–526 or DsRed–Monomer–FUS 165–526 or the corresponding DsRed control. The chromatin-bound proteins were prepared by SDS and benzonase extraction as shown in Fig. S1A. The chromatin-bound and soluble fractions were subjected to SDS/PAGE followed by Western blot. (C) DsRed2–FUS 165–526 showed a punctate distribution and nucleolar exclusion inside the nucleus, similar to that of the full-length FUS. The monomeric DsRed-tagged FUS 165–526 was evenly distributed in the nucleus. Cells were fixed 24 h after transfection and subjected to confocal microscopic analysis. Red, DsRed and DsRed-tagged FUS; blue, DAPI staining of DNA; and arrows, nucleoli. (D) Native gel electrophoresis of FUS in the chromatin-bound (CB) and soluble (S) fractions. The slow mobility of FUS suggests a high order assembly of FUS in the CB fraction.
Fig. 5.
Fig. 5.
RNA dependence of FUS self-assembly and chromatin binding. (A) FUS chromatin binding is dependent on RNA. HEK cell lysates were incubated with indicated amounts of RNase A for 20 min on ice before separation of the chromatin-bound and soluble fractions using the protocol as in Fig. S1A. The amount of chromatin-bound FUS in the presence of RNase A was examined by Western blot. (B) RNA dependence of chromatin binding of the DsRed2-tagged FUS 165–526. HEK cells were transfected with DsRed2–FUS 165–526 and harvested 48 h after transfection. Cell lysates were incubated with indicated amounts of RNase A for 20 min on ice and separated to the chromatin-bound and soluble fractions. The amount of chromatin-bound DsRed2–FUS 165–526 in the presence of RNase A was examined by Western blot. (C) RNA dependence of FUS self-assembly. The chromatin-bound fraction was incubated with indicated amounts of RNase A and subjected to native gel electrophoresis. The soluble fraction was included as a control.
Fig. 6.
Fig. 6.
Proposed model of FUS self-assembly and chromatin binding. Wild-type FUS forms high order assemblies and binds to active chromatin where FUS regulates gene transcription. FUS regulation of mRNA splicing does not require self-assembly or chromatin binding, thus is mediated by the pool of soluble FUS.
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