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aurelian/agents/goann/documents/Transcription_Factors_Annotation_Guidelines_Paper.md

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+Contents lists available at ScienceDirect
+
+BBA - Gene Regulatory Mechanisms
+
+journal homepage: www.elsevier.com/locate/bbagrm
+
+Gene Ontology representation for transcription factor functions
+
+Pascale Gaudet a, *, Colin Logie b, Ruth C. Lovering c, Martin Kuiper d, Astrid Lægreid e,
+Paul D. Thomas f
+a Swiss-Prot group, SIB Swiss Institute of Bioinformatics, 1 Rue Michel-Servet, 1211 Gen`eve, Switzerland
+b Molecular Biology Department, Faculty of Science, Radboud University, PO box 9101, 6500HB Nijmegen, the Netherlands
+c Functional Gene Annotation, Preclinical and Fundamental Science, UCL Institute of Cardiovascular Science, University College London, London, UK
+d Department of Biology, Norwegian University of Science and Technology, Trondheim, Norway
+e Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway
+f Division of Bioinformatics, Department of Preventive Medicine, University of Southern California, Los Angeles, CA, USA
+
+A R T I C L E  I N F O
+
+A B S T R A C T
+
+Keywords:
+Transcription
+Gene Ontology
+Biological databases
+Biocuration
+
+Transcription plays a central role in defining the identity and functionalities of cells, as well as in their responses
+to changes in the cellular environment. The Gene Ontology (GO) provides a rigorously defined set of concepts
+that describe the functions of gene products. A GO annotation is a statement about the function of a particular
+gene product, represented as an association between a gene product and the biological concept a GO term de-
+fines. Critically, each GO annotation is based on traceable scientific evidence. Here, we describe the different GO
+terms that are associated with proteins involved in transcription and its regulation, focusing on the standard of
+evidence  required  to  support  these  associations.  This  article  is  intended  to  help  users  of  GO  annotations  un-
+derstand how to interpret the annotations and can contribute to the consistency of GO annotations. We distin-
+guish between three classes of activities involved in transcription or directly regulating it - general transcription
+factors, DNA-binding transcription factors, and transcription co-regulators.
+
+1. Introduction
+
+The  Gene  Ontology  (GO)  develops  a  computational  model  of  bio-
+logical systems, ranging from the molecular to the organism level, across
+all species in the tree of life. GO aims to provide a comprehensive rep-
+resentation of the current scientific knowledge about the functions of
+gene products, namely, proteins and non-coding RNA molecules [1,2].
+GO is organized in three aspects. GO Molecular Functions (MF) describe
+activities that occur at the molecular level, such as “DNA binding tran-
+scription  factor  activity”  or  “histone  deacetylase  activity”.  Biological
+Processes (BP) represent the larger processes or ‘biological programs’
+accomplished by multiple molecular activities. Examples of broad bio-
+logical  process  terms  are  “transcription”  or  “signal  transduction”.
+Cellular Components (CC) are  the cellular structures in which a  gene
+product  performs  a  function,  either  cellular  compartments  (e.g.,  “nu-
+cleus”  or “chromatin”), or stable macromolecular complexes of which
+they are parts (e.g., “RNA polymerase II”). Together, annotations of a
+gene to terms from each of those aspects describe what specific function
+a gene product plays in a process and where this activity occurs in the
+
+cell. Ideally every gene product should have an annotation from each of
+the three aspects of GO.
+
+The specific genes expressed in a given cell define the identity and
+functionalities of that cell. Regulation of transcription is highly complex
+and leads to differential gene expression in specific cells or under spe-
+cific  conditions.  In  human  cells,  it  has  been  estimated  that  several
+thousand  proteins  participate  in  gene  expression  and  its  regulation,
+directly  or  indirectly  [3]  (Velthuijs  et  al.  BBAGRM-D-21-00020  this
+issue).  This  includes  the  general  transcription  machinery,  the  factors
+that make the chromatin more or less accessible, specific DNA-binding
+transcription factors, and the signaling molecules that regulate the ac-
+tivity  of  all  those  proteins.  This  complexity  is  difficult  to  accurately
+represent in ontological form. Tripathi et al. [4] redesigned that part of
+the  ontology  in  2013  to  define  precise  molecular  functions  for  the
+various proteins involved in transcription and its regulation. Nearly 10
+years  after  its  implementation,  we  had  to  acknowledge  that  this
+framework  was  too  complex  and  difficult  to  navigate,  leading  to
+inconsistent annotations and thus poorly serving the user community.
+The  work  described  here  was  also  motivated  by  the  https://www.gr
+
+* Corresponding author.
+
+E-mail address: pascale.gaudet@sib.swiss (P. Gaudet).
+
+https://doi.org/10.1016/j.bbagrm.2021.194752
+Received 6 January 2021; Received in revised form 24 August 2021; Accepted 25 August 2021
+
+BBA-GeneRegulatoryMechanisms1864(2021)194752Availableonline28August20211874-9399/©2021TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).P. Gaudet et al.
+
+Fig. 1. Transcription regulator activity branches of the Gene Ontology. (a) Graphical representation of the placement of the parent terms for transcription regulator
+molecular functions. Black headers correspond to MF and cyan headers to BP terms. (b) Transcription regulators are dbTF and coTFs. The general transcription
+initiation factors play a direct role in transcription. Top-level terms of each branch are highlighted in blue.
+
+eekc.org/  GREEKC  consortium,  whose  goals  include  curation  tools
+development,  reengineering  of  ontologies,  development  of  curation
+guidelines and text mining tools, developing platforms to analyze and
+render  the  molecular  logic  of  transcription  regulatory  networks  for
+which  a  robust  infrastructure  is  needed.  Therefore,  we  thoroughly
+reviewed  the  Gene  Ontology  representation  of  molecular  activities
+relevant to transcription, with a simpler and more pragmatic approach,
+more aligned with available experimental data.
+
+We  have  revised  the  GO  MF  terms  representing  the  activities  of
+proteins involved in transcription, with the input from domain experts.
+In addition to RNA polymerase, we defined three different types of ac-
+tivities that take place on the DNA to mediate or regulate transcription:
+general transcription factors (GTFs), DNA-binding transcription factors
+(dbTFs), and transcription coregulators (coTFs).
+
+Here we present the annotation approach recommended by the GO
+consortium  [5],  applied  to  the  recent  refactoring  of  the  transcription
+
+domain of GO. This approach aims to 1) help biocurators – annotation
+producers - interpret published data and correctly assign the MFs terms
+for GTF, dbTF, or coTF to a protein, and 2) help users understand how
+the  data  is  generated  and  how  to  interpret  them.  The  annotation  of
+factors  involved  in  transcription  and  its  regulation  is  challenging  for
+multiple  reasons.  Contrary  to  other  molecular  functions,  for  example
+enzymes, where one protein or a well-defined complex catalyses a pre-
+cise  reaction,  the  measurable  output  of  transcription  activities  is  the
+result of multiple nearly simultaneous activities of GTF, dbTF, coTF, as
+well  as  RNA  polymerase,  hence,  individual  activities  can  be  hard  to
+distinguish  experimentally.  Moreover,  these  factors  often  form  large
+complexes, such that the level of resolution of the experimental setup is
+essential to determine the precise activity of any given protein. Older
+experimental methods often did not provide enough details, leading to
+inaccurate classifications of certain proteins. In addition, researchers use
+“transcription factor” loosely, at times meaning GTF, dbTF, or coTF. This
+
+BBA-GeneRegulatoryMechanisms1864(2021)1947522P. Gaudet et al.
+
+Fig. 2. DNA binding branch of the Gene Ontology. This part of the Molecular Function (MF) ontology describes DNA binding. (a) Graphical representation of the
+placement of the terms describing sequence-specific promoter binding. (b) Hierarchical view of the sequence-specific transcription regulatory region binding terms.
+
+complicates the annotation process and necessitates solid expertise for
+correct interpretation of the data. The experimental data itself is difficult
+to parse for unambiguous assignment of a function to a protein: typi-
+cally, a single experiment is insufficient for accurately determining the
+function of these proteins, thus, interpretation of experimental results
+that investigate dbTFs must rely on pre-existing knowledge. Also, many
+proteins presumed to function as dbTFs have never been experimentally
+demonstrated to bind DNA, but their role is indirectly inferred by the
+presence  of  known  specific  DNA-binding  domains  and  in  some  cases,
+evidence of an effect on the transcription of putative direct target genes.
+To add to the complexity, the presence of a DNA-binding domain in a
+protein does not always imply that the protein functions as a dbTF [6].
+
+2. GO description of molecular functions relevant for
+transcription
+
+We  distinguish  between  three  types  of  activities  involved  in  tran-
+scription  or  directly  regulating  it:  general  transcription  factors
+(GO:0140223),  DNA-binding  transcription  factors  (GO:0003700),  and
+transcription co-regulators (GO:0003712). The general transcription
+initiation factor activity term and its descendants describe the activ-
+ities of general transcription initiation factors for RNA polymerase I, II
+and III, which play a direct role in the biological process of transcription
+at the core promoter (Sant et al., BBAGRM-D-21-00014 this issue). In
+contrast,  the  GO:0140110  transcription  regulator  activity  branch
+describes the activities of transcription regulators: dbTF and coTFs, that
+
+BBA-GeneRegulatoryMechanisms1864(2021)1947523P. Gaudet et al.
+
+act  at  any  type  of  cis-regulatory  module  (Fig.  1).  DNA-binding  tran-
+scription factors are adaptors that bind chromatin at specific genomic
+addresses to coordinately regulate the expression of genes sets. This is
+encoded in the ontology via links between the DNA-binding transcrip-
+tion  factor activity  term  and  its  descendants  and  to  their  counterpart
+branch of the MF ontology describing DNA binding. The GO:0000976
+transcription regulatory region sequence-specific DNA-binding sub-tree
+of GO includes terms describing specific regulatory regions, such as the
+core promoter (including the TATA box and the transcription start site),
+cis-regulatory  regions  (bound  by  dbTFs),  and  specific  types  of  cis-
+regulatory  motifs  (such  as  E-box  and  N-box).  An  overview  of  the  GO
+structure for DNA binding activities is shown in Fig. 2. The definitions
+and placement of GO terms in the ontology can be viewed in the AmiGO
+[7,8];  http://amigo.geneontology.org/amigo,  and  QuickGO  [9];
+https://www.ebi.ac.uk/QuickGO/ browsers.
+
+3. Strategy for annotating transcription-associated activities
+
+GO terms are associated with gene products based on two general
+approaches: from experimental data and from sequence inferences [10].
+The GO database has a total of 8 million annotations, about 7% of which
+are to human gene products. For human, there are >915,000 annota-
+tions derived from experimental data (GO release 2020-10-10 obtained
+from  http://amigo.geneontology.org).  Sequence  inference  methods
+provide more than 106,000 annotations for human proteins based on
+phylogenetic relationships (65,000 annotations) [11]; protein domains
+(6730  annotations)  [12];  and  Ensembl  orthology  predictions  (35,000
+annotations)  [13].  The  next  sections  describe  the  annotation  of  the
+different types of proteins involved in transcription and its regulation.
+
+3.1. Transcription activity annotations supported by experimental data2.
+
+The following annotation approach follows the recommendations of
+the GO consortium. First and foremost, it is necessary to use as much
+information as possible, rather than annotating articles individually and
+out of the wider context. When extracting information, a gene-by-gene
+or  pathway-by-pathway  approach  is  considered  best  practice  [5].
+Reviewing a range of articles ensures that the annotations closely reflect
+the current state of knowledge. Ideally, the corpus of annotations for a
+gene product should be based on multiple observations from different
+articles by independent research groups. Five steps used to determine
+whether  a  gene  can  be  annotated  as  a  transcriptional  regulator  are
+outlined  in  Fig.  3.  Appendix  1  provides  examples  of  each  of  those
+different activities.
+
+1.  Identify the starting hypothesis: are the authors characterizing
+a  transcription  regulator?  Scientific  models  are  built  by  adding
+new  data  to  the  existing  corpus  of  evidence.  New  data  can  either
+support  or  contradict  existing  models.  The  Introduction  section  of
+research articles can be used to understand what prior knowledge the
+article builds on, and which aspect of the existing model or what new
+model the authors are assessing. The hypothesis tested by the authors
+is essential to choose a GO term, with the caveat that inconsistent
+terminology  has  been  used  in  transcription  research  articles  and
+therefore may not always be aligned with the GO term categories.
+2.  Determine whether knowledge from specific protein domains or
+characterized orthologs support the hypothesis. The presence of
+specific domains and the existence of well-characterized orthologs
+can provide useful support for interpreting experimental data. Note
+that this data should be used with caution. For instance, ARID-, AT
+hook-, and some HMG-, GATA-, zinc finger domain-containing pro-
+teins and proteins binding structural features such as the DNA minor
+groove rarely bind DNA in a sequence-specific manner; some of them
+merely function to increase the avidity or stability of a transcription
+factor complex and its associated co-factors and do not - in their own
+capacity - provide the specific genomic address to guide transcription
+
+Fig.  3. Five  steps  to  transcription  activity  annotation.  The  five  key  steps  to
+associating a transcription MF term with a protein starts with identifying the
+starting hypothesis, to confirm that the authors are characterizing a GTF, dbTF
+or  coTF.  Secondly,  considering  whether  the  knowledge  from  specific  protein
+domains or characterized orthologs support the hypothesis. Thirdly, checking
+whether existing annotations from GO, UniProt and Model Organism databases
+are  consistent  with  the  hypothesis.  Fourthly,  reviewing  other  published
+experimental  data  to  ensure  no  contradictory  findings  have  been  reported.
+Finally, creating new GO annotations, if the experimental results are consistent
+with the identified hypothesis.
+
+to specified target genes. Such proteins are not considered dbTFs in
+GO.
+
+To support the association of a gene with a GO term from homolo-
+gous sequences from other species, only closely related orthologs whose
+function have been unambiguously characterized can be used if those are
+consistent with the experimental data presented in the article.
+
+-  GTFs  function  as  the  molecular  machine  that  assembles  with  the
+RNA polymerase at the promoter to form the pre-initiation complex
+(PIC).  GTFs  have  been  characterized  in  several  organisms,  from
+archaea  to  yeast  and  mammalian  cells  [14,15],  and  therefore
+orthology should provide strong support for the decision to associate
+these proteins with a child specific for RNA polymerase I, II or III of
+the  MF  term  “GO:0140223  general  transcription  initiation  factor
+activity”. In addition, the naming of GTFs is well established across
+human and model organism nomenclature groups and can be used to
+help guide these decisions. Thus, for human GTFs the HUGO Gene
+Nomenclature Committee (HGNC, www.genenames.org) provide the
+gene symbol TAF#, for TATA-box binding protein associated factors,
+and GTF2#s and GTF3#s, for general transcription factor II and III
+subunits respectively.
+
+- dbTFs are specific double-stranded DNA-binding transcription fac-
+tors that provide genomic addresses and respond to the conditions
+under which specific genes are expressed. Central to dbTF function is
+their  binding  to  specific  double-stranded  DNA  sequences  that  are
+often named transcription factor binding sites (TFBS). Gene products
+associated with the GO term “GO:0003700 DNA-binding transcrip-
+tion factor activity”  have the ability to bind DNA and this binding
+regulates the expression of a specific set of target genes. The direct
+target gene(s) can also be included in the annotation using the “has
+input  relation”.  A  human  dbTF  catalog  developed  by  the  GREEKC
+project  (  [6];  also  accessible  from  https://www.ebi.ac.uk/Qui
+ckGO/targetset/dbTF)  may  be  consulted  to  check  whether  a  spe-
+cific human protein is annotated to dbTF function with experimental
+or phylogenetic evidence. When considering proteins that belong to
+families of well characterized transcription factors, such as those that
+contain  bHLH,  bZIP,  homeobox,  ETS,  Forkhead,  etc.  domains  and
+proteins  with  a  one-to-one  ortholog  already  demonstrated  to  be  a
+
+BBA-GeneRegulatoryMechanisms1864(2021)1947524P. Gaudet et al.
+
+dbTF, then weaker evidence of DNA binding, such as ChIP experi-
+ments is sufficient. In contrast, special care must be taken to annotate
+proteins  bearing  domains  that  are  not  exclusively  found  in  tran-
+scription factors, such as RING, MYND and PhD zinc fingers. Simi-
+larly, for proteins with enzymatic activity: while there are rare cases
+of dbTFs with enzymatic activities, such as ENO1, dbTF and enzy-
+matic activity are usually mutually exclusive. For proteins not in the
+dbTF  catalog,  clear  experimental  or  phylogenetic  evidence  of
+sequence-specific DNA binding and gene transcription regulation via
+cognate DNA motifs located in gene-associated cis-regulatory mod-
+ules is required for the protein to be classified with high confidence
+as a dbTF.
+
+- coTFs: Transcription coregulators (also known as transcription co-
+factors; GO:0003712) represent a group of different functions that
+take place at cis-regulatory regions to make transcription of specific
+gene sets either more (coactivators) or less (corepressors) efficient.
+Coregulators  can  modify  chromatin  structure  through  covalent
+modification  of  histones,  ATP-dependent  chromatin  remodelling,
+and  modulate  dbTF  interactions  with  other  transcription  cor-
+egulators. We classify the Mediator Complex, which bridges dbTFs
+and  the  RNA  polymerase,  as  a  transcription  coactivator  [16–18].
+Many coTFs have enzymatic activity and normally exert their func-
+tion independent of high affinity binding to specific DNA sequences.
+CoTFs  that  do  bind  DNA  typically  recognize  very  short  DNA  se-
+quences  that  are  not  sufficiently  unique  in  the  genome  to  enable
+regulation of a limited set of genes in a discrete environmental or
+developmental stage. One example of this is CPF1, that binds the CpG
+dinucleotide and helps most CpG islands gain epigenomic marking
+[19–21].
+
+It  is  important  to  keep  in  mind  that  DNA  binding  proteins  that
+regulate transcription are not necessarily dbTFs. Key points that help
+distinguish between the three activities discussed above are that (i)
+dbTFs bind DNA in a sequence-specific manner, and regulate precise
+sets of genes; (ii) coTFs usually do not directly bind DNA, and when
+they  do  they  don't  exhibit  strong  sequence-specificity;  (iii)  coTFs
+often  have  catalytic  activities  (such  as  histone  methyltransferase,
+protein kinase, or ubiquitin ligase), which is highly unusual in dbTFs;
+(iv) GTFs are required for core promoter activity and are considered
+to act at each promoter to promote transcription initiation [14,22],
+although the exact subunit composition at individual promoters may
+vary.
+
+3. Confirm  that  existing  annotations  are  consistent  with  the  hy-
+pothesis. New annotations need to be consistent with existing an-
+notations, unless the existing annotations are believed to be wrong or
+out of date. Annotations made to a term as well as a more specific
+descendant reflect differences in granularity of annotation, and are
+not generally considered inconsistent. When the new annotation uses
+a term in a different branch than existing annotations, a review of the
+evidence supporting the existing annotations is undertaken and, if
+necessary, annotations that appear to be incorrect are disputed (see
+section “Ensuring a coherent set of annotations”).
+
+4.  Check  that  other  published  experimental  results  do  not
+contradict the hypothesis. The application of the gene-by-gene or
+pathway-by-pathway annotation approach ensures that results from
+other research articles are taken into account and that all annotations
+are  in  line  with  the  current  state  of  knowledge.  Again,  if  in-
+consistencies  are  noticed,  great  care  is  taken  to  confirm  correct
+interpretation  of the data, this is  particularly important if there is
+evidence for multiple, distinct transcription activity functions.
+5.  Validate that the experimental results are consistent with the
+hypothesis.  If  the  results  presented  in  the  curated  article  are
+consistent  with  the  hypothesis  presented  by  the  authors,  then  the
+appropriate transcription activity GO term(s) are associated with the
+gene product.
+
+Proteins that are  involved  in  transcription and  its  regulation  have
+historically  been  studied  through  small-scale,  focused  experimental
+approaches. For some examples of the small-scale experiments that do
+provide evidence for DNA binding transcription factor activity the bio-
+curator  can  use  Tables  3  and  4  of  Tripathi  et  al.  [4]  and  in  Santos-
+Zavaleta  et  al.  [23].  Recent  advances  in  high-throughput  methodolo-
+gies now provide robust data that, when interpreted with sufficient care,
+support the assignment of a function role to many proteins, including
+transcription  regulators.  This  includes  HT-SELEX  [24,25],  Protein
+Binding Microarrays [26], ChIP [27], one- and two-hybrid experiments
+[28,29]. For these experiments, the data quality and the false positive
+rate  must  be  evaluated  before  annotations  are  created.  For  example,
+human HT-SELEX data will have more false positives if native dbTFs are
+assayed  in  nuclear  extracts  or  over-expressed  in  eukaryotic  cells,
+compared with heterologous proteins purified from prokaryotic cells, as
+the latter reduces the probability of indirect interactions with endoge-
+nous factors. For high-throughput transcription data, only articles with
+low rates of false positives, are curated. Those various techniques pro-
+vide  multiple  independent  lines  of  evidence,  strengthening  the  confi-
+dence  in  the  annotation  when  they  converge  on  a  single  motif  or
+molecular  function.  The  GO  recommendations  on  curation  of  high-
+throughput experimental data should be applied when such data is an-
+notated [30].
+
+3.2. Annotations based on non-experimental evidence
+
+There are only about 500 human dbTFs for which there is experi-
+mental evidence satisfying the criteria presented here. Across all areas of
+biology several reliable methods infer protein function from available
+experimental data. Indeed, there are approximately 1000 human pro-
+teins annotated as dbTFs by non-experimental methods (Lovering et al.
+same BBA issue, prepublication available at [6]). Phylogenetic annota-
+tions  are  assigned  by  a  group  of  biocurators  with  expertise  in  evolu-
+tionary  biology,  and  require  experimental  evidence  for  at  least  one
+member  of  a  clade  of  evolutionarily  related  proteins  [11].  The  GO
+knowledgebase also contains GO terms assigned by automated pipelines
+based  on  protein  domain  (InterPro2GO)  and  orthology  (Ensembl).
+InterPro2GO [12] is based primarily on local (partial) homology: pro-
+tein domains are mapped to specific GO terms, and any protein with one
+of  these  domains  will  be  annotated  to  the  appropriate  GO  term(s).
+Ensembl Compara [13] generates groups of one-to-one orthologs among
+closely related species and propagates all experimental annotations to
+each members of the group. While manual annotations based on these
+methods are allowed, the GO consortium recommends using the auto-
+mated pipelines that are maintained centrally and ensure a consistent
+annotation corpus across all annotated species.
+
+4. Ensuring a coherent set of annotations
+
+During the process of annotation other relevant annotations associ-
+ated with the gene are reviewed. If there are conflicting annotations, the
+supporting data should be reassessed to determine whether the anno-
+tations  are  inconsistent  with  the  data,  in  which  case  the  annotations
+must be fixed [5].
+
+In cases where the primary data is conflicting across different articles
+(for example a protein is sometimes described as a transcription factor,
+and sometimes as a coregulator), then the literature will be reviewed
+carefully to decide whether the annotation is incorrect (bad choice of
+term, wrong protein annotated), whether the knowledge has evolved, if
+the protein plays multiple roles under different conditions (i.e., acts as a
+DNA-binding transcription factor in certain contexts and as a cofactor in
+others). If no activity has yet been established, no MF annotation will be
+made.
+
+Note  that  individual  DNA-binding  transcription  factors  can  act  as
+both activators or repressors dependent on the context, hence associa-
+tion of both activator and repressor terms with a single protein is not
+
+BBA-GeneRegulatoryMechanisms1864(2021)1947525P. Gaudet et al.
+
+Fig. 4. Representation of biological context of dbTF activity. The level of cyclin-dependent kinase inhibitor p21 (CDKN1A) is regulated by the transcription factor
+p53 (TP53) upon DNA damage, signaling cell cycle arrest to the cell (http://noctua.berkeleybop.org/editor/graph/gomodel:5fa76ad400000000).
+
+considered inconsistent. The specific conditions under which this hap-
+pens, such as relevant signaling pathways, cell type, as well as specific
+target genes, etc., may be further specified through additional context
+details ([31]; see an example of a GO-CAM model in Fig. 4).
+
+5. Pitfalls in annotating transcription regulators
+
+During the review of dbTF GO annotations [6], in which over 3000
+GO  annotations  were  reviewed,  a  variety  of  common  errors  in  data
+interpretation  were  identified.  One  of  the  most  common  errors  was
+caused  by  the  difficulty  in  distinguishing  a  dbTF  from  a  coTF,  as  the
+evidence for those two functions can be quite similar. To prevent this
+error, biocurators ensure that the protein has a sequence-specific dou-
+ble-stranded DNA-binding domain and conduct an exhaustive review of
+the  literature,  including  articles  associated  with  the  protein's  close
+orthologs. Furthermore, the literature supporting the dbTF activity of a
+protein that also has evidence for another function, in particular, RNA
+binding, will be carefully checked before assigning a dbTF activity. The
+work on the human dbTF catalog added a GO ‘DNA-binding transcrip-
+tion factor activity’ annotation to 583 proteins, and removed erronous
+assignments for 256 proteins (Lovering et al. BBAGRM-D-20-00141 this
+issue).
+
+Transcription  regulators  most  often  act  as  members  of  complexes,
+some of which also contain proteins with other activities. In some cases,
+only some subunits of a complex interact with DNA: for instance, while
+the RFX complex contains three members: RFX5, RFXAP and RFXANK,
+only  RFX5  binds  DNA  directly.  But  the  DNA-binding  ability  of  the
+complex is facilitated by all three subunits so RFXAP and RFXANK are
+not coTFs [32]. In this case, RFXAP and RFXANK are annotated using the
+“contributes to” qualifier, to indicate that they participate in, but are not
+directly responsible for the activity.
+
+Another  activity  that  can  easily  be  confused  for  a  coTF  is  a  dbTF
+inhibitor. These proteins interact with a dbTF, but not at the DNA, to
+prevent  the  dbTF  from  reaching  its  target  genes.  Well  characterized
+examples  are  the  I-SMADs,  SMAD6  and  SMAD7  [33],  that  act  by
+competing  with  active  SMADs  at  receptors,  thus  blocking  further
+intracellular signaling, and should be annotated to “GO:0140416 tran-
+scription regulator inhibitor activity”.
+
+It must be noted that these approaches to avoid errors in dbTF ac-
+tivity assignment are not unequivocal, as some proteins do have multiple
+functions. For example, the glucocorticoid receptor (NR3C1), which is a
+
+canonical dbTF, has recently been shown to bind double-stranded RNA
+motifs  [34];  ATF2  (activating  transcription  factor  2)  and  CLOCK  are
+dbTFs that have been reported to also exhibit histone acetyltransferase
+activity  [35–38];  some  dbTFs,  such  as  NFIB  (nuclear  factor  I  B),  also
+function as dbTF inhibitors [39]. Finally, general and sequence-specific
+effects can be difficult to separate, as has been established for the MYC
+dbTF [40].
+
+6. Conclusion
+
+The  annotation  approach  presented  here  is  designed  to  help  bio-
+curators annotate factors involved in transcription and its regulation, as
+well as for users of GO annotations to understand their meaning and the
+evidence behind them. This work complements the redesign of this part
+of  the  GO  to  significantly  simplify  the  ontology  structure.  The  new
+ontology structure and the present standards were applied to the review
+of human proteins associated with GO terms describing dbTF activity
+[6].  We  anticipate  that  adoption  of  this  annotation  approach  by  all
+groups  who  produce  GO  associations  will  increase  annotation  consis-
+tency across all species, for transcription and also more widely across all
+areas represented by GO.
+
+Declaration of competing interest
+
+The authors declare that they have no known competing financial
+interests or personal relationships that could have appeared to influence
+the work reported in this paper.
+
+Acknowledgements
+
+We  thank  many  GREEKC  and  GO  consortium  members  for  useful
+discussions that led to the development of these guidelines, in particular
+Marcio L. Acencio, Helen Attrill, and Valerie Wood.
+
+Funding sources
+
+The  GO  Consortium  is  funded  by  the  National  Human  Genome
+Research  Institute  (US  National  Institutes  of  Health),  grant  number
+HG002273. RCL has been supported by Alzheimer's Research UK grant
+(ARUK-NAS2017A-1)  and  the  National  Institute  for  Health  Research
+University  College  London  Hospitals  Biomedical  Research  Centre.
+
+BBA-GeneRegulatoryMechanisms1864(2021)1947526P. Gaudet et al.
+
+GREEKC is supported by the COST Action grant CA15205.
+
+Appendices. Supplementary data
+
+Supplementary data to this article can be found online at https://doi.
+
+org/10.1016/j.bbagrm.2021.194752.
+
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