Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305-5329, USA
Author for correspondence (e-mail:
fuller{at}cmgm.stanford.edu)
Accepted 17 June 2004
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SUMMARY |
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Key words: TAF, TFIID, Drosophila, Spermatogenesis, Spermatocyte, Transcription
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Introduction |
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A striking example of a tissue-specific TAF homolog required for execution
of a developmentally regulated transcriptional program appears during
differentiation of male germ cells in Drosophila
(Hiller et al., 2001). The
cannonball gene (can) of Drosophila, which encodes
a homolog of dTAF5 expressed only in male germ cells, is required for normal
transcription in primary spermatocytes of a set of spermatid differentiation
genes required for normal spermatogenesis
(Hiller et al., 2001
;
Lin et al., 1996
;
White-Cooper et al., 1998
).
Flies null mutant for can are viable and female fertile but male
sterile. The requirement for can function is gene selective: only a
specific set of genes normally expressed in wild-type primary spermatocytes
are affected, while a number of other genes are transcribed normally in
spermatocytes from can mutant males. While it is now clear that
cell-type-specific TAF homologs such as can and mTAF4b can play
important roles in tissue-specific gene expression, the mechanisms by which
they function at specific promoters are not understood. To identify proteins
that might collaborate with the dTAF5 homolog can to regulate
expression of specific target genes in Drosophila spermatocytes, we
investigated the expression and function of other TAFII homologs in
the Drosophila genome.
We show that Drosophila primary spermatocytes express several additional tissue-specific TAF homologs that act to control expression of spermatid differentiation genes: nht (homolog of dTAF4), mia (homolog of dTAF6), sa (homolog of dTAF8), and rye (homolog of dTAF12). Mutations in nht, sa and mia cause the same phenotypes as can, blocking meiotic cell cycle progression and spermatid differentiation, and have similar effects on transcription in primary spermatocytes of several target genes involved in spermatid differentiation. The nht and rye proteins interact structurally when co-expressed in bacteria, like their generally expressed homologs TAF4 and TAF12. Strikingly, the structural interaction was tissue-specific: nht did not interact with dTAF12, and dTAF4 did not interact with rye in the bacterial co-expression assay. We propose that these five Drosophila testis-specific TAF homologs collaborate in an alternative TAF-containing protein complex expressed in primary spermatocytes to regulate expression of a set of genes involved in spermatid differentiation.
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Materials and methods |
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Molecular cloning and sequence analysis of nht, mia, sa and rye
cDNAs corresponding to mia, nht and rye transcripts were
isolated by screening a testis cDNA ZAP library provided by T.
Hazelrigg (Columbia University) or the BDGP AT library
(http://www.fruitfly.org/EST/index.html).
cDNA corresponding to the sa coding region was obtained by RT-PCR. A
79-base 5' UTR for sa and a possible 5' end of the mRNA
for nht and rye were identified from testis
poly(A)+ mRNA using the 5' GeneRacer system (Invitrogen). No
different forms of the mia predicted protein coding region were
apparent from the ESTs in the BDGP database, the two testis cDNA clones
characterized, or RT-PCR of fragments of the mia ORF from testis mRNA
compared to mRNA from 0-24-hour-old embryos. Primers for RT-PCR corresponded
to base pairs 956-977 and bp 1805-1825 of the cDNA, residing in exon 1 and 3,
respectively. RT-PCR using embryo RNA gave an 870 bp product, the expected
size for the fully processed mia transcript target, as well as a 1320
bp product expected if unprocessed mia transcript was the template.
No products were generated in control reactions lacking RT.
The mia genomic rescue construct contained a 6.1 kb
EagI-XhoI genomic fragment from cosmid 80E1 (Clare O'Connor,
Boston College) cloned into the NotI-XhoI sites of pCaSpeR-4
(Rubin and Spradling, 1983).
The approximately 10.5 kb nht genomic rescue construct D7 is
described by Yagi and Hayashi (Yagi and
Hayashi, 1997
). A 3.2 kb genomic DNA containing 1 kb both upstream
and downstream of the sa coding sequence obtained by PCR from wild
type genomic DNA was cloned into pCasper-4 and introduced into flies by
P-element transformation. The mia, nht or sa genomic
transgenes rescued the respective mutant males to fertility and restored
normal spermatogenesis, based on phase contrast microscopy of testis squash
preparations.
RT-PCR to assess tissue-specific expression of sa was performed using Qiagen Onestep RT-PCR kit with primers corresponding to the first 18 and the last 18 nt of the predicted protein coding sequence producing an 825 bp fragment amplified from male RNA. Point mutations in the nht, mia and sa alleles were identified by sequencing bulk PCR products amplified from genomic DNA of homozygous mutant flies using gene-specific oligonucleotides. cDNA and PCR sequences were aligned and analyzed using Sequencher (Gene Codes Corp.) and MacVector (Oxford Molecular Group plc) DNA analysis software.
The Nht, Mia, Sa and Rye protein sequences, predicted from the cDNA sequences, were used to search nucleotide sequence databases translated in all reading frames (tBLASTn). The cDNA sequences were AY752877(nht), AY752878(mia), AY752879(sa) and AY752880(rye).
Alignments of nht, rye, mia and sa predicted proteins to
the histone fold domains of the generally expressed TAFs were constructed
using HMMER release 2.3 (Durbin et al.,
1998) against the PFAM database V.10.0
(Bateman et al., 2002
). These
domains were used to seed multiple sequence alignments performed using
ClustalW (Thompson et al.,
1994
) and the T-Coffee multiple sequences alignment method
(Notredame et al., 2000
). The
multiple alignments were performed through stepwise, independent TAF and
histone family alignments then by aligning separate profiles of each family.
With the highly divergent sequences, T-Coffee was found to provide much more
reliable alignments than ClustalW, and was used in the alignments presented in
this paper. Alignment coloring and percentage identity/similarity for pairwise
comparisons were obtained using GeneDoc
(Nicholas et al., 1997
).
Protein alignments were refined and threaded onto existing x-ray crystal
structures using the Swiss-PDB Viewer software package and Swiss-Model server
(http://www.expasy.org/swissmod/SWISS-MODEL.html)
(Guex and Peitsch, 1997).
Putative structures of nht and rye were threaded onto 1H3O.
Protein alignments were modified using the Sippl-like threading energy
minimization functions in Swiss-PDB Viewer and resubmitted to the Swiss-Model
server until optimal RMSD was obtained. An average noise cut-off RMSD of 0.87A
was obtained by threading and averaging the RMSD of three gap-free
helix-loop-helix proteins unrelated to the TAF family. As negative controls,
we also aligned, as oligomers, Nht-TAF12 and TAF4-Rye.
In situ hybridization and RNA and DNA blot analysis
In situ hybridization and DNA/RNA blot analysis were as described
previously (Hiller et al.,
2001). mRNA from adult testis or 0- to 24-hour embryos was
isolated by homogenization in TRIzol Reagent (Life Technologies) followed by
isolation of mRNA using Micro-FastTrack 2.0 (Invitrogen). RT-PCR of
mia was carried out using Qiagen OneStep RT-PCR kit. For northern
blots and in situ hybridization, the mia probe was generated from a
DNA fragment corresponding to the first 1331 bp of the mia cDNA, the
nht probe was generated from a plasmid containing a PCR product
corresponding to bp 89-789 of the cDNA, and the rye probe was
generated from a plasmid containing PCR sequences corresponding to genomic
DNA, including intron sequences, between bp 16 and 438 of the cDNA. For
sa, in situ hybridization was performed with antisense and sense
probes of the entire protein coding sequence. Other probes for in situ
hybridization have been described previously
(White-Cooper et al., 1998
;
Hiller et al., 2001
).
Tests of physical interaction in bacteria
The GST-Nht fusion protein was produced by introducing EcoRI
(5' region coresponding to amino acid 5) and HindIII
(immediately 3' to the natural stop codon) sites into sequences flanking
the Nht coding region and cloning that region into vector pGEX-KG
(Guan and Dixon, 1991). A
similar region of dTAF4, amino acid 647 through the natural stop codon, was
also cloned into pGEX-KG. A portion of the Rye protein was expressed from
pACYC184-11b by cloning a PCR product encoding amino acid 65 to the natural
stop codon. Primers used to generate the PCR product were designed to
introduce a Met residue preceeding the first Ile (amino acid 65) of the Rye
sequence, followed by Pro Tyr Phe Ser Pro Tyr Gln. A similar dTAF12 fusion
protein expressed amino acids 91-169. A linker containing the FLAG epitope
(Met Tyr Lys Asp Asp Asp Asp Lys Ala Ala Ala) was inserted before the first
Met (converted NdeI site) of the Rye and dTAF12 fusion proteins. All
constructs were sequenced to insure that no unintended changes were introduced
by PCR and fusion joints were in-frame.
Co-expression of histone-fold-containing fusion proteins made use of
expression vectors pGEX-KG (Guan and
Dixon, 1991) to generate appropriate GST fusion proteins, and
pACYC184-11b (Fribourg et al.,
2001
), which contains a p15A origin of replication compatible with
the ColE1 origin of replication on pGEX-KG, to generate FLAG-tagged proteins.
Plasmids were transformed into bacterial strain BL21, and grown at 26°C in
medium containing 100 µg/ml ampicillin and 35 µg/ml chloramphenicol to
an A600 of 0.5. Expression of fusion proteins was induced with 0.3 mM
isopropyl ß-D-thiogalactoside (IPTG) and followed by growth overnight at
16°C. GST-fusion proteins were purified using glutathione-Sepharose 4B
(Pharmacia Biotech, Inc) per the manufacturer's instructions. Proteins were
separated by SDS-PAGE and transfered to nitrocellulose membrane (Hybond ECL,
Amersham Pharmacia Biotech). Blots were probed with anti-GST (Amersham
Pharmacia) at a dilution of 1:1000 and anti-FLAG M1 (Sigma) at 1:666. In our
studies, as also shown by several other groups working with HFD TAFs, proteins
with a histone-fold motif were often not soluble in the absence of their
proper binding partner. As purification by binding to glutathione-Sepharose
requires the GST fusion proteins to be stable and soluble, insoluble proteins
were not detected in our binding assays. The GST-TAF4 fusion protein fragment
we tested was slightly soluble when expressed alone and could be detected upon
longer exposures. The TAF12-FLAG fusion protein we tested was stable, but did
not bind and elute from the glutathione beads in the absence of its TAF4
HFD-containing binding partner. Solubility and co-purification of FLAG-Rye on
glutathione-Sepharose was only observed in the presence of GST-Nht. Likewise,
purification of FLAG-TAF12 on glutathione-Sepharose was only observed in the
presence of GST-TAF4.
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Results |
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The testis TAF homologs mediate a common gene expression program in spermatocytes
To assess the role of the TAF homologs in vivo, we identified null mutant
alleles. Two nht mutants were identified by screening a large
collection of male sterile lines (Wakimoto
et al., 2004) for mutants with a meiotic arrest phenotype similar
to can. The mutation causing the nht meiotic arrest
phenotype was localized to polytene region 35C by recombination mapping and
deficiency complementation (Materials and methods). As this region contained
CG15259, the dTAF4 homolog, we sequenced genomic DNA amplified from CG15259 by
PCR. Both nht alleles carried base changes from the background
chromosome that caused premature stop codons in the predicted CG15259 open
reading frame (Table 2). A
transgene with a 10.5 kb fragment of genomic DNA containing CG15259 fully
rescued the spermatocyte arrest phenotype and male sterility of
nhtz-5347/Df. Together these results established
that nht encodes the TAF4 homolog
(Table 1).
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The sa locus was previously mapped to polytene chromosome interval
78C9-D2 (Lin et al., 1996).
Additional local deletions made by mobilization of the EP(3)0339
P-element insert (Materials and methods) further localized sa
proximal to EP(3)0339. The sa gene was found to lie in a
large intron of CG6014, transcribed from the opposite strand. Sequencing of
genomic DNA amplified from the two sa alleles by PCR reveled a 414 bp
deletion and 158 bp insertion in the predicted protein coding region in
sa1, which had been recovered from the wild as
VO45 by D. Lindsley in the Rome screen
(Sandler et al., 1968
), and a
C to T transition resulting in a premature stop codon in the EMS-induced
sa2 compared to its background chromosome
(Table 2). A 3.2 kb genomic DNA
fragment containing CG11308 fully rescued the spermatocyte arrest and male
sterility phenotypes of sa1/sa2 males
when introduced into flies by P-element-mediated germline transformation,
establishing that sa corresponds to CG11308
(Table 1).
Mutations in nht, mia or sa caused the same meiotic
arrest phenotype as loss of function of the testis-specific dTAF5 homolog
can. Testes from nht males contained cells at the earliest
stages of spermatogenesis up through primary spermatocytes, but germ cells
failed to initiate meiotic cell division and the testes filled with mature
primary spermatocytes (Fig.
2B). No stages of spermatid differentiation were detected in
nht testes and mature spermatocytes eventually degenerated at the
testis base. Loss of function of mia or sa caused a similar
meiotic arrest phenotype, with mature spermatocytes arrested at the G2/M
transition of meiosis I and lack of spermatid differentiation
(Lin et al., 1996).
Strikingly, although mia mRNA expression was not specific to the
adult male germline, mia function appeared to be required mainly for
spermatogenesis: mia1/miaz-3348 flies
were viable and female fertile.
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Several testis TAFs are homologs of general TAFs containing a histone fold domain motif
The full-length protein encoded by nht was homologous to the
C-terminal portion of dTAF4 and its human homolog hTAF4, but lacked the
characteristic N terminal glutamine rich domain
(Fig. 3A). A 924 bp cDNA
representing the nht transcript isolated from a testis cDNA library
and confirmed by RT-PCR from testis mRNA closely matched the 1 kb size
predicted by northern analysis. Sequencing of this near full length
nht cDNA revealed an open reading frame encoding a predicted 245
amino acid protein. Comparison with the genomic DNA sequence revealed no
introns in the region covered by the cDNA. The initial methionine was preceded
by in frame stop codons in the cDNA, suggesting that it represented the start
of the protein. The yeast homolog Mpt1p (yTAF4) also does not contain the
extended N-terminal glutamine rich domain.
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The predicted protein encoded by rye is homologous to TAF12, the histone fold motif-containing binding partner of TAF4. A 550 bp rye cDNA isolated from a testis cDNA library closely matched the 600 bp transcript size estimated from northern blots (Fig. 1). Sequencing of this near full length rye cDNA revealed an open reading frame encoding a 138aa predicted protein with homology to dTAF12. An in frame stop codon located six base pairs upstream of the predicted initial methionine in the cDNA suggested that this corresponded to the start of the protein.
The predicted Rye protein contained a histone fold domain region (amino acid residues 68-130 of rye) that aligned well with the histone fold domain of TAF12 and histone H2B (Fig. 3C,D). The sequence of the generally expressed dTAF12 as aligned in Fig. 3D threaded onto the crystal structure of hTAF12 in the hTAF4:hTAF12 dimer well, with an RMSD of 0.09A for the region from aa91-aa164. Although rye was more diverged, the rye HFD sequence aligned as in Fig. 3D also fit onto the hTAF4:hTAF12 dimer crystal structure (RMSD=0.47). Again, rye and the TAF12 proteins from C. elegans to humans showed a striking pattern of alternating hydrophobic and charged or polar residues echoing the similar pattern in histone H2B in the region of the predicted HFD, especially in the second helix (Fig. 3D).
The mia gene encodes a protein homologous to dTAF6. A 2400 bp cDNA isolated from a testis cDNA library corresponded in size to the mia transcript expressed in testis estimated from northern blots. The testis mia cDNA also contained almost 600 bp of 5' untranslated sequence containing multiple stop codons in all three reading frames upstream of the predicted initial methionine.
The mia N-terminal region resembled corresponding regions of the
generally expressed dTAF6 and TAF6 from other organisms, although conservation
at the level of amino acid sequence was relatively low
(Table 3). However, multiple
sequence alignments using PFAM domain alignments and T-Coffee (see Materials
and methods) revealed that amino acid residues 35-94 of the predicted Mia
protein aligned with the histone fold domain of TAF6 and histone H4 with
respect to the pattern of hydrophobic, charged and polar residues
(Fig. 3F). Conservation of this
pattern was strongest in the region extending from the middle of the alpha 2
helix through loop 2 and the alpha 3 helix of the HFD, based on the
dTAF6:dTAF9 crystal structure (Xie et al.,
1996). In the nucleosome, the regions of alpha 2, loop 2 and alpha
3 of histone H4 are involved both in dimerization, involving residues internal
to the H3:H4 pair, and in forming non-symmetrical tetrameric bonds between H4
and H2B via exposed residues (Luger et
al., 1997
). The sequence of Mia as aligned in
Fig. 3F threaded onto the
crystal structure of dTAF6 in the dTAF6:dTAF9 heterodimer with an RMSD of 0.79
for aa33-aa94. In addition to the N-terminal predicted HFD, the Mia protein
contained an extended central region (aa176-437 of Mia) with a pattern of
hydrophobic, charged, polar and proline residues conserved in TAF6 proteins
from yeast to humans. Embedded in this `TAF6 domain' were several regions of
significant amino acid sequence conservation
(Table 3) between the predicted
Mia protein, dTAF6 and TAF6 from other organisms
(Fig. 3F).
The sa gene encodes a protein homologous to dTAF8, also known as Prodos. Sequencing of an 825 bp sa cDNA isolated by RT-PCR from testis mRNA revealed three protein coding exons separated by two introns (FBgn0037080). The cDNA had an in frame stop codon 84 base pairs upstream of the predicted initial methionine.
The N-terminal region of sa resembled the corresponding regions of
the generally expressed dTAF8 (Prodos) and TAF8 from other organisms.
Although conservation at the level of amino acid sequence was relatively low
(Table 3), domain searches
(PFAM) and multiple sequence alignments using T-Coffee (see Materials and
methods) suggested that amino acid residues 4-70 of the predicted Sa protein
contained a predicted histone fold domain, similar to TAF8 homologs from a
variety of species with respect to the pattern of hydrophobic, charged and
polar residues (Fig. 3H). Conservation of this pattern was strongest in the predicted alpha 2 helix
region. Although no crystal structure was yet available for TAF8 homologs,
secondary structure predictions by the Psipred program also suggested an
-helix loop
-helix loop
-helix pattern in this region of the predicted sa protein,
consistent with a histone fold domain structure
(Fig. 3H, green bars). In
support of the prediction of an N-terminal histone fold domain in dTAF8,
deletion of amino acids 1-39 from the generally expressed Drosophila
TAF8 homolog Prodos disrupted binding between dTAF8 (Prodos)
and its binding partner dTAF10B
(Hernandez-Hernandez and Ferrus,
2001
).
The predicted Sa protein also contained an extended central region (aa107-186) with significant amino acid sequence conservation (Table 3) and a pattern of hydrophobic, charged, polar and proline residues conserved in TAF8 homologs from yeast to humans (Fig. 3H). This region is embedded in a larger domain (aa78-186) listed in the pFam database as pFamB-10670, an unannotated domain characteristic of this family of TAFs (Fig. 3H, blue).
Two testis-specific TAF homologs with histone fold domains interact as testis-specific binding partners
Because TAF4 and TAF12 physically interact as binding partners through
their histone fold domains (Gangloff et
al., 2000; Werten et al.,
2002
; Yokomori et al.,
1993
), we tested whether the Drosophila testis-specific
TAF4 and TAF12 homologs nht and rye also interact
structurally, using a bacterial co-expression and GST pulldown assay
(Fig. 4). In bacteria carrying
a GST-nht fusion construct encoding full length Nht and an empty
vector in place of a Rye-FLAG fusion construct, the GST-nht fusion
protein was detected in total bacterial extracts (T) under inducing
conditions, but was not soluble in the absence of Rye-FLAG fusion protein
(Fig. 4, lane pair 1). A
portion of Rye containing the histone fold region fused to a FLAG epitope was
not stable and failed to accumulate when expressed in bacteria in the absence
of Nht (Fig. 4, lane pair 2).
However, when both the Nht and Rye fusion proteins were co-expressed in the
same bacteria, both the GST-Nht fusion protein and the FLAG-tagged Rye
HFD-containing fragment accumulated in the total bacterial extract (T) and
were soluble. When the GST-Nht fusion protein was isolated from extracts from
bacteria expressing both the Nht and Rye fusion proteins by binding to
glutathione-Sepharose, the FLAG-Rye fusion bound and co-eluted with GST-Nht
(Fig. 4, lane 3B).
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Expression of the testis TAF mRNAs is mutually independent
To investigate whether the testis TAFs are transcribed independently at the
onset of the primary spermatocyte program or whether some of the testis TAFs
might regulate mRNA expression of the others, we assayed mRNA expression of
nht, can, mia, sa and rye in spermatocytes from males mutant
for nht, can, mia or sa by in situ hybridization to
whole-mount testes. In all cases examined, mRNA for the testis TAFs
accumulated in the various mutant spermatocytes
(Fig. 5;
Table 4). At times, the testis
TAF transcripts appeared sharply at the boundary between spermatogonia and
spermatocytes (Fig. 5), as in
wild type. However, in some cases staining for the transcript appeared
gradually in spermatocytes further from the testis apical tip. We could
distinguish no clear pattern among these variations, which may be due in part
to the probe or the degree to which spermatocytes are less crowded up into the
testis apical third in the absence of differentiating spermatids in the
mutants. Notably, for the alleles examined, can mRNA accumulated in
can mutant spermatocytes. The same was true for nht, sa, and
mia mRNA in nht, sa, or mia mutant spermatocytes,
respectively (Fig. 5B,G;
Table 4), indicating that
transcription of any particular testis TAF did not depend on wild-type
function of the respective protein itself. Transcripts for can, mia, sa,
nht and rye also accumulated in spermatocytes mutant for
aly (Fig. 5E,H), which
has meiotic arrest and spermatid differentiation mutant phenotypes similar to
the can class testis TAFs, although transcripts appeared to
accumulate gradually rather than turn on abruptly in early spermatocytes.
Accumulation of aly transcripts or protein was previously shown to be
independent of wild-type function of the testis TAFs can, mia and
sa (White-Cooper et al.,
2000).
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Discussion |
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If the five testis TAFs identified to date act in a multi-subunit complex resembling TFIID, then the identity of the other subunits expected in a TFIID-like complex is a puzzle. The five TAF homologs we have identified may substitute for their more generally expressed homologs in a chimeric TFIID complex, where the other components are the generally expressed TAFs. Alternatively, the Drosophila testis TAFs may participate in a TFIID-like complex where the other components are highly diverged from their more generally expressed forms. We note that the nht, can, mia, sa, and rye predicted proteins are more diverged from their generally expressed homologs than the generally expressed Drosophila homologs are diverged from their human counterparts. Another possibility is that the testis TAF homologs act in a different type of protein complex, for example a HAT complex or with the Polycomb complex of chromatin modifying factors, to regulate the cell-type-specific expression of terminal differentiation genes required for spermatogenesis.
The testis TAF4 homolog nht lacked the long, glutamine-rich N
terminus characteristic of both the generally expressed Drosophila
homolog and TAF4 from mammals. This glutamine-rich N-terminal domain can
interact structurally with certain transcriptional activator proteins in vitro
and has been proposed to help TFIID mediate activated transcription, perhaps
by tethering TFIID to transcriptional activators bound at enhancers (reviewed
by Hochheimer and Tjian,
2003). If so, then the testis-specific homolog nht may
render a possible testis-specific TFIID-like complex less sensitive to
transcriptional activators that might normally interact through the glutamine
rich N-terminal domain of dTAF4.
Northern blot analysis suggested low levels of alternate transcript forms
of mia expressed in females and embryos. Alternate transcripts have
also been described for TAF6 in human cells
(Bell et al., 2001). However,
analysis of null mutants, including an allele with an early stop codon in the
mia open reading frame, suggested that wild-type function of
mia is required for spermatogenesis but not for female fertility or
embryonic development. It is possible that the mia protein is only
expressed in spermatocytes, or that the generally expressed homolog dTAF6 can
substitute for mia function in other tissues. Perhaps mia
protein may be required to allow nht:rye to bind into a
testis-specific TFIID or HAT-like complex, so that mia function is
essential only where it is necessary to incorporate nht:rye. The HFD
containing dTAF6 forms a heterodimer partner with dTAF9
(Xie et al., 1996
). However,
searches of the Drosophila genome have not yet revealed an obvious
candidate for a second homolog of dTAF9 that might serve as a binding partner
with mia.
The generally expressed TAF8 homolog Prodos binds specifically to
dTAF10B in vitro and in yeast two-hybrid assays. The Drosophila
genome encodes two homologs of TAF10. However, unlike the testis-specific TAF
homologs we describe here, both dTAF10 and dTAF10B are expressed during
embryogenesis, with some degree of tissue specificity
(Georgieva et al., 2000).
Preliminary tests in the bacterial co-expression assay did not reveal
interaction between the testis-specific dTAF8 homolog sa and either
dTAF10 or dTAF10B (X.C., unpublished data). Searches of the
Drosophila genome have not yet revealed an obvious candidate for an
additional dTAF10 homolog that might serve as an HFD heterodimer partner with
sa.
Tissue-specific GTFs and the male germline
Several tissue-specific TAFs and other GTF component homologs have been
found to be expressed in the testis in differentiating male germ cells in
mammals as well as in Drosophila. Altering the composition of the
general transcription machinery may be particularly important for gene
expression in the male germline. Differentiation of male gametes in both
Drosophila and mammals depends on a robust germline-specific
transcriptional program (Fuller,
1993; Goldberg,
1996
; Hecht,
1993
). Expression of many genes required for spermatid
differentiation takes place in spermatocytes in Drosophila and in
spermatocytes and/or early round spermatids in mammals. Many genes that are
transcribed in somatic cells at other stages of development are expressed in
male germ cells from testis-specific promoters. In addition, a number of
generally expressed genes in Drosophila have homologs that are only
or mainly expressed in the testis. In several cases the cis-acting regulatory
sequences that drive expression of the testis-specific transcripts have been
shown to be contained within short regions positioned near the start of
transcription. Humans express a testis-enriched subunit of TFIIA and a
testis-specific TAF1 homolog (Ozer et al.,
2000
; Upadhyaya et al.,
1999
; Wang and Page,
2002
) while a testis-specific homolog of TAF7 has been identified
in mouse (Pointud et al.,
2003
). In addition, wild-type function of the TATA-binding protein
homolog TRF2 is required in mouse to produce mature sperm
(Martianov et al., 2001
;
Zhang et al., 2001
). Both our
studies of the testis TAFs of Drosophila and studies of knockout
mutant mice lacking mTRF2 function indicate that these testis-specific
homologs of GTF components are required for normal transcription and terminal
differentiation in male germ cells. It is possible that chromatin may be in a
different condensation state in spermatocytes and early round haploid
spermatids than in many somatic cells and so may require specialized forms of
the general transcription machinery to recognize or access testis-specific
promoters within an altered chromatin landscape.
One striking finding was the tissue specificity of the structural
interactions between dTAF4:dTAF12 compared with the nht:rye proteins.
Examination of the virtual structure of the histone fold domains of
nht and rye threaded onto the crystal structure of the
hTAF4:hTAF12 HFD heterodimer did not reveal any obvious single reason for the
specificity of the dimer partners observed in biochemical assays, suggesting
that the specificity of the binding partner interaction may be the result of
an additive effect of a number of residue interactions across the HFD. One
notable difference between the predicted structures of dTAF4:dTAF12 compared
with nht:rye, based on threading onto the hTAF4:hTAF12 HFD crystal
structure, appeared in the region where loop1 of TAF4 interacts with loop2 of
TAF12. Loop 1 of hTAF4 and loop 2 of hTAF12 both have a short region of beta
sheet, which interacts in parallel to anchor the end of the 2 helix
interaction in the hTAF4:hTAF12 crystal structure
(Werten et al., 2002
). The
dTAF4:dTAF12 sequences fit well across this region, yielding predicted
interacting beta sheets based on the threading algorithm used (SwissModel).
However, the nht:rye predicted structures did not fit this region
well. Both nht loop 1 and rye loop 2 lacked a predicted
short beta sheet in the virtual heterodimer formed by threading onto the
hTAF4:hTAF12 crystal structure. When we took the virtual structures of dTAF4
and rye predicted by threading and placed them in the heterodimer
positions, an acidic clash between Asp732 of the dTAF4 HFD and Asp117 of
rye was created in the region where loop1 of TAF4 would contact loop
2 of rye. A second notable difference in the predicted heterodimers
of dTAF4:dTAF12 and nht:rye was that the nht
His-75:rye Ser-104 interaction contained a hydrogen bond. This
interaction would be expected to be much stronger than the weak Ala-Val
interaction at the corresponding position in TAF4:TAF12. This interaction
would also be abrogated in an nht:TAF12 or TAF4:rye
heterodimer. However, the alpha-1 helix of TAF4, corresponding to the region
where nht His-75 is located, was not required for TAF4-TAF12
dimerization in yeast TAF4 mutant rescue experiments
(Thuault et al., 2002
). The
dTAF4:dTAF12 structure predicted by threading on to the hTAF4:hTAF12 crystal
structure is likely to be relatively reliable (RMSD=0.14A). However, the amino
acid sequences of the predicted HFD motifs of nht and rye
were considerably diverged from the corresponding regions of both the human
and the generally expressed Drosophila TAF4 and TAF12 proteins
(Table 3). As a result, the
virtual structure of the nht:rye HFD heterodimer, calculated from
threading, is much less reliable (RMSD=0.74A) and may underestimate the
divergence of the protein structures from the heterodimer between the
generally expressed homologs. Since the nht:rye heterodimer can be
stably expressed in bacteria (Fig.
4) the best way to compare the structures and probe the molecular
basis of the specificity between the generally expressed and the
testis-specific predicted heterodimers may be through solving the crystal
structure of the testis-specific nht:rye complex.
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ACKNOWLEDGMENTS |
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Footnotes |
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