dataesr/scientific-paragraphs-categorization huggingface dataset:
Except for some rare exceptions  [20] , MHD-only TFs have not been confirmed experimentally and there is some debate about whether the MHD can act independently. For example, in all experimentally proven TFs listed in the TRANSFAC database (https://genexplain. com/transfac/, accessed on 3 September 2022), the MHD is always located downstream of a DBD  [5]. A recent genome-wide study of the complement of TF in the fungus Aspergillus nidulans revealed numerous discrepancies between the predicted protein sequences and the deduced sequences from experimental transcriptomic data, with approximately 30% of the TFs needing some type of correction  [21]. Among the badly predicted TFs, a large majority (78%) concerns the Zn2C6-and/or MHD-containing sequences which frequently exhibit non-predicted or non-processed introns leading to premature stop codons and erroneous sequences.
It is interesting that most of the A. nidulans MHD-only proteins have a domain with predicted DNA binding (mainly of the Zn2C6 type) after an RNA sequence analysis. These high-throughput experimental results prompted us to reassess the fungal-specific MHD-containing TF family, by developing a domain-centric error-tracking approach that takes into account potential mispredictions of protein sequences. As a starting point, we collected proteins containing an MHD from three sequence databases with different levels of human expertise involved in the genome annotation process. First, the Saccharomyces Genome Database (SGD) is dedicated to the budding yeast S. cerevisiae  [22]  and provides comprehensive information including protein sequences from a collection of 48 S. cerevisiae strain genomes.
Second, the UniProtKB/Swiss-Prot database is the expertly curated component of UniProtKB  [23]. Third, the UniProtKB/TrEMBL database contains computer-generated annotations for all translations of the EMBL nucleotide sequence entries. We then focused our analysis on the MHD-only TFs by applying a specific error-tracking protocol that uses different DBD-MHD combinations to identify potentially mispredicted genes in available fungal genomic sequences, and especially mispredictions that affected the protein domain organization. Our large-scale analysis of almost 17,000 MHD-only TFs allowed us to verify that at least 90% of them possess upstream genomic sequence regions coding for a DBD, mostly of the Zn2C6 type. These results suggest that the vast majority of the MHD-only TF sequences present in public databases result from errors, and that the Zn2C6-MHD domain pair represents a canonical domain signature defining the most predominant family of TFs composed of two fungal-specific domains.
Collection of SGD Sequences The 44 proteins from the S. cerevisiae S288C strain with a Zn2C6-MHD domain organization  [12]  were identified in the SGD database (Table  S1 ), and their annotated orthologs in the 47 available strains (Table  S2 ) were downloaded from the SGD website (http://sgd-archive. yeastgenome. org/sequence/strains/strain_alignments. tar, accessed on 27 April 2022). Genome assemblies for the 47 strains were also downloaded from the SGD website (http://sgd-archive. yeastgenome. org/sequence/strains, accessed on 27 April 2022). Ortholog sequences that did not contain the conserved CX{2}CX{6}CX{5,16}CX{2}CX{6,8}C motif were considered to be potentially erroneous. For each potentially erroneous sequence, we performed a TBLASTN alignment of the S288C reference protein sequence with the corresponding genome assembly.
Collection of UniprotKB Sequences Fungal proteins were identified in the UniProt 2022_01 database  [23]  by querying for proteins annotated with the InterPro entry IPR007219: Transcription_factor_dom_fun or Fun-gal_trans, which covers the middle homology domain (MHD) specific to these transcription factors. Domain architectures of all proteins containing an MHD were then extracted from the InterPro v86. 0 database  [19]. The 37,646 UniProt sequences annotated with an MHD, but no DBD, were considered to be potentially erroneous (MHD-only). Potentially erroneous sequences from the reviewed UniProt/Swiss-Prot entries and unreviewed UniProt/TrEmbl entries were processed separately: the 12 UniProt/Swiss-Prot proteins with no DBD were analyzed manually, while the 37,634 UniProt/TrEMBL sequences were input to the error identification protocol (Figure  1 ) and described in detail in the following sections.
Construction of BLAST Databases of Proteins with Full Domain Architecture UniProt sequences containing an MHD (IPR007219) in combination with a DBD were used to construct BLAST reference databases. Two BLAST databases were constructed: one for each of the two main DBD types, namely Zn2C6 (IPR001138) and C2H2 (IPR013087), found in this TF family and to which the well-studied GAL4 protein belongs. The Zn2C6 BLAST reference database contained 80,456 sequences, while the C2H2 BLAST reference database contained 6314 sequences.
Extraction of Genomic Sequences For all potentially erroneous sequences in UniProt, the corresponding genomic DNA sequences were extracted from the Ensembl database  [24] , when an Ensembl cross-reference was available in the UniProt database. To improve detection of the missing DBD, the full-length gene sequences were retrieved together with an additional 1000 nucleotides upstream of the 5′ end of the gene. For the 37,634 potential error sequences, 16,760 genomic DNA sequences were found in the Ensembl database.
Extraction of Genomic Sequences For all potentially erroneous sequences in UniProt, the corresponding genomic DNA sequences were extracted from the Ensembl database  [24] , when an Ensembl cross-reference was available in the UniProt database. To improve detection of the missing DBD, the full-length gene sequences were retrieved together with an additional 1000 nucleotides upstream of the 5 end of the gene. For the 37,634 potential error sequences, 16,760 genomic DNA sequences were found in the Ensembl database.
Identification of Nearest-Neighbor Reference Sequences For each MHD-only sequence, BLASTP searches were performed in the two BLAST reference databases containing Zn2C6 and C2H2 sequences in combination with an MHD. The nearest-neighbor sequences with the required domain combination (i. e. , DBD and MHD) were selected if a BLASTP hit was identified with an E-value < 0. 005. Figure  2  shows the E-value distribution of BLASTP hits obtained for all neighbor sequence searches. For each MHD-only sequence, BLASTP searches were performed in the two BLAST reference databases containing Zn2C6 and C2H2 sequences in combination with an MHD. The nearest-neighbor sequences with the required domain combination (i. e. , DBD and MHD) were selected if a BLASTP hit was identified with an E-value < 0. 005. Figure  2  shows the E-value distribution of BLASTP hits obtained for all neighbor sequence searches.
Identification of Missing DBD Sequences For each MHD-only sequence with a BLASTP hit to a nearest-neighbor reference protein, two complementary approaches were implemented to search for the missing DBD sequence. First, a local TBLASTN search was performed in the genomic sequence of the potential error sequence, using the protein DBD sequence segment of the nearest neighbor as a query. TBLASTN alignments with an E-value < 0. 0001 were taken into account. Second, a global pairwise alignment was performed between the genomic sequence of the MHD-only sequence and the full-length protein sequence of the nearest neighbor, using the ProSplign software developed by the NCBI (https://www. ncbi. nlm. nih. gov/sutils/static/prosplign/prosplign. html, accessed on 12 August 2022). ProSplign is a tool for protein-to-genomic sequence alignment, and it is an integral component of the NCBI Eukaryotic Genome Annotation Pipeline.
Identification of Missing DBD Sequences For each MHD-only sequence with a BLASTP hit to a nearest-neighbor reference protein, two complementary approaches were implemented to search for the missing DBD sequence. First, a local TBLASTN search was performed in the genomic sequence of the potential error sequence, using the protein DBD sequence segment of the nearest neighbor as a query. TBLASTN alignments with an E-value < 0. 0001 were taken into account. Second, a global pairwise alignment was performed between the genomic sequence of the MHD-only sequence and the full-length protein sequence of the nearest neighbor, using the ProSplign software developed by the NCBI (https://www. ncbi. nlm. nih. gov/ sutils/static/prosplign/prosplign. html, accessed on 12 August 2022). ProSplign is a tool for protein-to-genomic sequence alignment, and it is an integral component of the NCBI Eukaryotic Genome Annotation Pipeline.
Genes are first localized on the genomic sequence in a compartmentalization step that starts with computing protein-to-genomic blast hits. These give initial insight into the structure of compartments. Hits are separated into two same-strand sets and then compartments are identified within each strand. To do so, the optimization problem is formally defined in terms of genomic sequence coverage and then solved with a dynamic programming algorithm. ProSplign has been shown to produce accurate spliced alignments and is able to compute alignments of distantly related proteins with low similarity. Pairwise alignments obtained from TBLASTN and ProSplign were analyzed to identify potential DBD-encoding sequence segments in the erroneous sequences. Finally, the potential DBD-encoding sequence segments were compared to an HMM representing the DBD downloaded from the Pfam protein family database  [25] : PF00172 for the Zn2C6 DBD and PF00096 for the C2H2 DBD.
To do this, the hhmsearch program from the HMMER suite  [26]  was used and sequences with an E-value < 0. 1 were considered as hits. In addition to the hmmsearch E-value and to eliminate a number of false positive hits, we also checked for conserved amino acids: for Zn2C6 DBD, two occurrences of the pattern C-x(2)-C (where x is any amino acid) were required, while for C2H2 DBD, one occurrence for each of the C-x(2,4)-C and H-x(3,5)-H patterns was required. In order to determine whether our sequence curation protocol over-predicted DBDs in the potentially erroneous sequences, we used the same error identification protocol to search for Zn2C6 DBD in the C-terminal region of the proteins (i. e. , downstream of the MHD). The results of this analysis are described in the Supplementary Methods.
MHD-Containing Proteins in the SGD Database We first analyzed the MHD-containing proteins in the SGD: a database that provides comprehensive integrated information for S. cerevisiae. The reference (S288C) strain of S. cerevisiae contains 44 proteins with an MHD (Table  S1 ), with all of them exhibiting a Zn2C6-MHD domain pair  [12]. In addition to S288C, the SGD contains genome assemblies and annotations for a further 47 strains of S. cerevisiae (Table  S2 ). For each of these 47 strains, we extracted the annotated orthologs of the 44 S288C Zn2C6-MHD proteins. If all orthologs were conserved in all strains, we would expect a total of 2068 orthologs (44 orthologs from each of the 47 strains), but only 1793 orthologous sequences were found in the SGD (Table  1 ). In other words, 275 (13%) orthologous sequences were not predicted.
These included the nine gene sequences split over multiple scaffolds, which could not be resolved due to the genome assembly issues, and one sequence with a frameshift error (although a manual analysis of this genome sequence indicated a frameshift error affecting one of the conserved cysteines in the Zn2C6 domain). In order to validate our predictions for the 253 erroneous sequences, we searched for RNA-seq datasets in the NCBI Gene Expression Omnibus (GEO) project corresponding to the different S. cerevisiae strains, focusing on the strains with the highest number of potentially mispredicted genes. This transcriptome analysis is described in Supplementary Methods. For each mispredicted gene with a coverage of at least 30 reads, the aligned reads in the region of the gene were manually reviewed, confirming that all the predicted DBD sequences were expressed at similar levels to the MHD portion (Figure  S1 ).
MHD-Containing Proteins in the UniProt Database We then queried the UniProt database for all proteins annotated with the MHD (Inter-Pro ID: IPR007219), resulting in a total of 126,861 proteins, with 126,691 in the unreviewed TrEMBL section and 170 in the reviewed Swiss-Prot section. The MHD-containing proteins had a wide range of domain architectures, with 1905 different architectures listed in the InterPro database, although the most frequent domain pairs were as follows: (i) MHD with a Zn2C6 DBD (IPR001138), (ii) MHD-only, and (iii) MHD with one or two C2H2 DBD (IPR013087), as shown in Figure  3  and Table  S4. segments of a single genome scaffold with a sequence identity of at least 95%, although the matching segments were found in different reading frames. These frameshifts were mainly due to the insertion of one or two bases in the genome sequence of the S.
MHD-Containing Proteins in the UniProt Database We then queried the UniProt database for all proteins annotated with the MHD (In-terPro ID: IPR007219), resulting in a total of 126,861 proteins, with 126,691 in the unreviewed TrEMBL section and 170 in the reviewed Swiss-Prot section. The MHD-containing proteins had a wide range of domain architectures, with 1905 different architectures listed in the InterPro database, although the most frequent domain pairs were as follows: (i) MHD with a Zn2C6 DBD (IPR001138), (ii) MHD-only, and (iii) MHD with one or two C2H2 DBD (IPR013087), as shown in Figure  3  and Table  S4. For the 170 proteins from the Swiss-Prot section, nearly 90% contained the Zn2C6-MHD domain pair, although this combination was found in a smaller proportion of the TrEmbl proteins with only 63.
Manual Analysis of the 12 Swiss-Prot MHD-Only Sequences Since Swiss-Prot entries are curated by experts, we manually investigated the twelve MHD-only sequences in this database (Table  S5 ). Where possible, we extracted the corresponding genome sequence from either ENSEMBL  [24]  or GENBANK  [27]  databases and translated the genome sequence in the three frames to search for potential DBD encoding regions. This was not possible for four of the twelve sequences. For Q5AR44 and A0A5C1RF03, the gene was located at the start of a contig and the genome region upstream of the annotated gene was not available. For B8NJG5, according to the ENSEMBL database, the upstream gene coded for a small protein coding a Zn2C6 DBD. Finally, A6SSW6 was a shorter protein of length 179 (compared to >500 for the other Swiss-Prot proteins), and the MHD hit in the InterPro database was a partial domain.
Automatic Analysis of 16,760 TrEMBL MHD-Only Sequences Based on the manual analysis of Swiss-Prot described in the previous section, an automatic protocol was developed to analyze potentially erroneous sequences retrieved from the UniProt/TrEMBL database. The first step in the protocol involved identifying the corresponding genomic sequences in the ENSEMBL database. This resulted in a set of 16,760 sequences that were used as input for the main error detection step (see Methods). Two different methods were implemented to locate genomic regions within or upstream of the gene that could encode the missing DBD, using either a local or global alignment approach. Figure  5  shows the number of DBD identified by the two methods. J. Fungi 2023, 9, 424 9 of 16 cysteines/histidines corresponding to potential zinc binding amino acids highlighted in blue or green.
Automatic Analysis of 16,760 TrEMBL MHD-Only Sequences Based on the manual analysis of Swiss-Prot described in the previous section, an automatic protocol was developed to analyze potentially erroneous sequences retrieved from the UniProt/TrEMBL database. The first step in the protocol involved identifying the corresponding genomic sequences in the ENSEMBL database. This resulted in a set of 16,760 sequences that were used as input for the main error detection step (see Methods). Two different methods were implemented to locate genomic regions within or upstream of the gene that could encode the missing DBD, using either a local or global alignment approach. Figure  5  shows the number of DBD identified by the two methods. By integrating the results of the local and global alignment searches, DBD sequences could be proposed for 14,482 (86%) of the 16,760 MHD-only sequences tested (Table  S6 ).
Reassessment of Domain Pairs in MHD-Containing Sequences If the missing DBD sequences proposed here were integrated into the public databases, the number of MHD-only proteins would be reduced from 12 to 4 for Swiss-Prot, and from 16,760 to 2278 for TrEMBL (Figure  6  and Table  S6 ). More importantly perhaps, this would lead to a significant difference in the distribution of domain pairs present in MHDcontaining sequences. In the public databases, this distribution was 65%, 5%, and 30% for Zn2C6-MHD, C2H2-MHD, and MHD-only, respectively. However, our error-tracking protocol indicated that the true distribution was closer to 90%, 6%, and 4% for Zn2C6-MHD, C2H2-MHD, and MHD-only, respectively. sequences (69. 4%) were strictly identical to the DBD sequences detected by Chang and Ehrlich, thus highlighting the accuracy of our automatic error-checking protocol.
Reassessment of Domain Pairs in MHD-Containing Sequences If the missing DBD sequences proposed here were integrated into the public databases, the number of MHD-only proteins would be reduced from 12 to 4 for Swiss-Prot, and from 16,760 to 2278 for TrEMBL (Figure  6  and Table  S6 ). More importantly perhaps, this would lead to a significant difference in the distribution of domain pairs present in MHD-containing sequences. In the public databases, this distribution was 65%, 5%, and 30% for Zn2C6-MHD, C2H2-MHD, and MHD-only, respectively. However, our errortracking protocol indicated that the true distribution was closer to 90%, 6%, and 4% for Zn2C6-MHD, C2H2-MHD, and MHD-only, respectively. Concerning the phylogenetic distribution of the DBD-MHD domain pair, at the phylum level, MHD domains are present in all phyla except Microsporidia and Cryptomycota (Figure  S2 ).
Despite this overall enrichment of C2H2 in the basidiomycete TF, within the MHD sequences, the proportions of ZN2C6 and C2H2 are similar in both clades (90% and 7% for Ascomycota compared to 92% and 3% for Basidiomycota) (Table  S7 ). The level of protein sequence errors is of course dependent on the quality of the genome sequencing, assembly, and annotation. A small number of well-characterized organisms had no MHD-only sequences in the UniProt database, including model organisms such as S. cerevisiae, Yarrowia lipolytica, or Ustilago maydis. Nevertheless, some model organisms had a small number of MHD-only sequences, for example, Schizosaccharomyces pombe had twenty-seven proteins annotated with an MHD, of which two proteins had no or Candida albicans with 28 MHD-containing proteins, of which only one had no DBD: Q5AJ63_CANAL (Figure  7 ).
A small number of well-characterized organisms had no MHD-only sequences in the UniProt database, including model organisms such as S. cerevisiae, Yarrowia lipolytica, or Ustilago maydis. Nevertheless, some model organisms had a small number of MHD-only sequences, for example, Schizosaccharomyces pombe had twenty-seven proteins annotated with an MHD, of which two proteins had no DBD, or Candida albicans with 28 MHD-containing proteins, of which only one had no DBD: Q5AJ63_CANAL (Figure  7 ). At the other extreme, Rhizopus delemar had thirty-four MHD-containing proteins of which only six were also annotated with a DBD, i. e. , 82% were MHD-only proteins. According to our protocol, DBD could be detected for a further twenty-four MHD proteins and only four (12%) lacked a DBD. A further manual analysis of these four MHD-only proteins showed that two genes (I1BR31, I1C782) had regions coding complete Zn2C6 domains within 1500 nt upstream of the 5′ end, while one gene (I1CF81) had a partial hit within the default threshold of 1000 nt upstream of the 5′ end (-490 to -192).
According to the public databases, I1CF81 was coded by a gene with one exon and contained one known domain: the MHD. The partial DBD hit for I1CF81 in fact contained a misprediction of a short exon coding for two amino acids, as shown in Figure  8. At the other extreme, Rhizopus delemar had thirty-four MHD-containing proteins of which only six were also annotated with a DBD, i. e. , 82% were MHD-only proteins. According to our protocol, DBD could be detected for a further twenty-four MHD proteins and only four (12%) lacked a DBD. A further manual analysis of these four MHD-only proteins showed that two genes (I1BR31, I1C782) had regions coding complete Zn2C6 domains within 1500 nt upstream of the 5 end, while one gene (I1CF81) had a partial hit within the default threshold of 1000 nt upstream of the 5 end (-490 to -192).
According to the public databases, I1CF81 was coded by a gene with one exon and contained one known domain: the MHD. The partial DBD hit for I1CF81 in fact contained a misprediction of a short exon coding for two amino acids, as shown in Figure  8. of this domain pair. In contrast, the C2H2-MHD domain pair is found only in Dikarya (Basidiomycota and Ascomycota). Interestingly, it has been shown previously that there is a significant difference in the TF repertoire of ascomycete and basidiomycete fungi  [8] , and in particular that the Zn2C6 family (33%) is much more prevalent than the C2H2 (10%) in ascomycete TF, compared to basidiomycete TF (20% and 15% for Zn2C6 and C2H2, respectively). Despite this overall enrichment of C2H2 in the basidiomycete TF, within the MHD sequences, the proportions of ZN2C6 and C2H2 are similar in both clades (90% and 7% for Ascomycota compared to 92% and 3% for Basidiomycota) (Table  S7 ).
The level of protein sequence errors is of course dependent on the quality of the genome sequencing, assembly, and annotation. A small number of well-characterized organisms had no MHD-only sequences in the UniProt database, including model organisms such as S. cerevisiae, Yarrowia lipolytica, or Ustilago maydis. Nevertheless, some model organisms had a small number of MHD-only sequences, for example, Schizosaccharomyces pombe had twenty-seven proteins annotated with an MHD, of which two proteins had no DBD, or Candida albicans with 28 MHD-containing proteins, of which only one had no DBD: Q5AJ63_CANAL (Figure  7 ). At the other extreme, Rhizopus delemar had thirty-four MHD-containing proteins of which only six were also annotated with a DBD, i. e. , 82% were MHD-only proteins. According to our protocol, DBD could be detected for a further twenty-four MHD proteins and only four (12%) lacked a DBD.
A further manual analysis of these four MHD-only proteins showed that two genes (I1BR31, I1C782) had regions coding complete Zn2C6 domains within 1500 nt upstream of the 5′ end, while one gene (I1CF81) had a partial hit within the default threshold of 1000 nt upstream of the 5′ end (-490 to -192). According to the public databases, I1CF81 was coded by a gene with one exon and contained one known domain: the MHD. The partial DBD hit for I1CF81 in fact contained a misprediction of a short exon coding for two amino acids, as shown in Figure  8.
Discussion In this work, we used a domain-centric in silico approach to show that the second most abundant fungal-specific TF family in the public databases, namely the MHD-only TFs, results largely from genome annotation errors leading to unpredicted DBD. Taking advantage of the high-quality sequences of the S. cerevisiae reference strain S288C and the availability of numerous other fungal genomes, we defined an error-tracking strategy involving increasing levels of difficulty: starting with the analysis of the MHD-only sequences present in 48 closely related S. cerevisiae strains, followed by the MHD-only sequences in the expert curated Swiss-Prot database, and finally, in the automatically generated TrEMBL database. The reference S. cerevisiae S288C strain has no MHD-only TF coding genes and we showed that, for 95% of the MHD-only TF genes observed in the other S.