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Gene Interaction Studies

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Welcome back to the course of Plant Developmental Biology. We are still discussing about theMolecular Genetics approaches for the studying Plant Development. So, if I recall previousclass, we were discussing about the reverse genetics based approaches to study a particulardevelopmental processes in plant.And, we have covered how to identify a particular gene, how to choose a particular gene to studyits function, then how to analyse its differential as well as temporal and spatial expressionpattern at a particular stage of the development or in a particular organ and tissue, thenwe have taken this gene for the functional study which two approaches mainly we havestudied one is the gain of function approach and the loss of function approach.Now, so we have a gene, we have its function, we know what kind of phenotype this gene haswhen we make, when we down regulate this gene or when we silence this gene. And, secondthing we have a phenotype or we know what happens if we ectopically over express thisgene. Now, the next part what is remaining in this one is that, if you have a gene whatis the next and next will be the gene interaction study or the mechanism, what is the mode ofaction, how a particular gene is regulating a particular process this is important.So, another thing that one particular developmental process is not usually regulated by a singlegene. So, you might end up identifying multiple genes which might having similar phenotypeor same phenotype, then how to study, what kind of interaction, what kind ofrelationshipall those genes have for a particular in a particular developmental process.The first thing is that let us assume that if you have two genes and if you have mutantloss of function mutant of both the genes and if you find same phenotype, if you seethat in both the mutants phenotype is same then there is a question is the first thingis that is the both mutants in the same gene or in the different gene. How to rule outthis possibility? This you can do by genetically and you can study the genetic interactionyou can make if I take one example. So, if one mutant which is m1 and anothermutant which is m2 both are having same phenotype both are recessive loss of function mutantsand let us assume the phenotype is short root, if I may make a cross and if both the mutationsare in the same locus, what is going to happen? You know that the genes exist and there aretwo loci and if these mutants are in the same gene then you can expect that somewhere hereis the mutation. Here the site of mutation might be different,but in the same gene, but if you make a cross and go to the F1 generation, what are yougoing to have? You are going to have one allele from one mutant and another allele from anothermutants, but in both the cases, but in this heterozygous F1 plant both the alleles aredisrupted. So, you are going to have F1 plants with mutantphenotype. If this is the case, then you can say that these both independent mutants therethe mutation site is in the same gene probably in the same gene. On the other hand, if inthis mutant mutation is let us assume in gene A and there is another gene B which is alsoplaying role in the same developmental process. And in second mutant if mutations in geneB, but both are homozygous when you cross them and go to the F1 generation you are goingto have gene A disrupted from here, but you will have allele of A as a normal from anotherplant. Here you will have gene B allele normal fromthis plant, but here you are going to have B interrupted, but in this in both in thisheterozygotes what will happen that the wild type copy both the wild type copies of thegene will complement the mutant phenotype which means that in F1 you are not going toget any phenotype. So, this kind of genetic analysis you perform then you can tell thatwhether both the mutants are in the same gene or in the different gene, then another kindof analysis. So, basically here what we are trying to discusstoday is that what are the different types of interactions. The first interaction wecan call is the genetic interaction if genetically two mutants or two genes are interacting,what does it mean? It means that if there are two mutants or two genes which are regulatingsame developmental pathway, let us take example if both are regulating root length then they,are they working in the same pathway? Are they working in the different pathways? thiswe can solve by having a genetic analysis by doing performing genetic analysis, howyou can do? You can make a kind of combinatorial mutants, you can combine both the mutant andlook the phenotype we will see the example. Another things that you can identify suppressorsor enhancers of the mutants. So, you take a mutant and then again mutagenize and tryto find another double mutants where either the previous mutant phenotype is enhancedor suppressed and then you establish their interaction through the genetic analysis.So, these kinds of interactions are called genetic interactions. Then second thing isthat if they are the genetic interacting or they may be interacting, may not be interactingthe next question is come, are they physically also interacting?So, there are lot of transcription factor, lot of developmental regulators they are knownto interact directly protein-protein interaction and they can make higher order complex andthat complex is actually regulating the process. So, how to study protein-protein interaction,that would be another thing and to establish, if you have more than one regulator. Secondthing is this is particularly protein DNA interactions or RNA interaction this dependslet us assume that if your developmental regulator is a transcription factor, which means thatit will go and bind to some DNA element to activate gene expression or for its function.So, to establish so there could be two kinds of genes, one genes which your transcriptionfactor is directly regulating, it is going physically binding to the promoter or thecis-regulatory elements of your gene and activating or repressing the expression depending onits nature or it is indirectly regulating. In indirect case your transcription factorX is regulating gene A and then gene A is regulating gene B.So, if you take X with respect to X, B is also regulated by X, but X is not directlyregulating B, it is indirectly regulating B. And, then you can based on this kind ofgenetic regulatory physical interaction, you can build and you can understand what is theregulatory network foundation. You can also take help of the co expression analysis. So,if you check a particular tissue or particular developmental stage and identify what arethe genes which are co expressed, you can basically predict that there might be interactionbetween them, if both are present in the same cell or in the same tissues.So, we will take one by one. So, first start with the genetic interaction so genetic interactions.So, as I said that, that you can do by identifying either modifiers which could be either suppressoror enhancers. One possibility is that if you have a mutation let us assume you have a mutantwhere mutation is in this particular gene, but if you take and try to identify a suppressormutant, if you mutagenesis again there is again possibility that another mutants whichhappens in the same gene either at the same site or at the different site and the secondmutants might restore the function of this particular gene such kind of mutants are calledintragenic suppressor. In another possibility is that you have amutant, you have a phenotype now second mutation you identify in a totally independent or differentgene and if this mutation is suppressing phenotype of this mutants then it is called extragenic.So, you can identify extragenic mutation, you can establish their genetic interaction,you can establish their physical interaction, you can establish their regulatory interactionand try to understand how what is the mode of action.When we are analysing whether they are working in the same pathway or different pathway,if they are working in the different pathway then then it means be that both there aremore than one parallel pathways going on and both the parallel pathways they are regulatingthe same biological process, but if they are working in the same pathway then you haveto establish which gene is upstream which gene is downstream is A regulating B or Bregulating A. So, which gene is upstream and which geneis downstream and how you can establish this? Again through the genetic interaction andthe common way of doing it that you make a different mutants, higher order mutants, combinatorialmutants, double mutant, triple mutant and then analyze. Genetic redundancy we will takeseparately how to study this.So, I will take some of the example this is a well studied case of a floral or floraltransitions. So, basically what happens in the plant at up to a certain time point, plantundergo the process of vegetative growth then it decide to transition, but the transitionfrom vegetative phase to reproductive phase is a very important process in the plant toensure proper sexual reproduction or successful sexual reproduction.Therefore, it is regulated by multi multiple regulators, multiple pathways and, but thesepathways some of them are working in independent manner and then there are interdependent manner,they are there is a huge amount of crosstalk. But how this crosstalk was established forexample, if you look here there are four pathways photoperiod, autonomous, vernalization andgibberellic acid pathway and all these pathways, this is a very simple picture, but there thispathways are quite complex and all they are eventually regulating the transition floraltransition; so, I will take few of the example and if you look.So, if you look the photoperiod, photoperiod means that the amount of light a plant isgetting based on that we can define entire plant into three category either they arelong day plant, short day plant, or day neutral plant. So, in long day condition, what happens?One gene which is getting activated is CONSTANST and then CONSTANST activates two pathway,one is FT and SOC1 and FT and SOC1, they are having their independent or parallel pathwaysto regulate the floral transition, how to establish this? If you look this mutant phenotype.So, here the total number of leaves is counted when there is a transition occurs if you havemore leaves then it is delayed flowering if you have less leaf then it is early flowering.So, if you look a typical wild type plant. So, wild type plants they have usually approximately15 leaf before they make 15 rosette leaves before they transit from vegetative phaseto reproductive phase, but if you look this ft mutants, they are making almost 40 leavesbefore the transition which means that there is a huge delay in the flowering, but if youif you over express CONSTANST if you increase the amount of CONSTANST, you can see thatthere is early flowering. So, even less than 10 leaves are made andplant has already undergone the process of flowering, but another interesting thing whatyou have seen. So, this tells that FT is activator of both are activators of the flowering, butwhat happens if you take CONSTANST over expression in the ft-10, ft mutant background the CONSTANSTcannot induce the flowering which means that CONSTANST is important, CONSTANST is importantin activating the flowering, but it is working through FT.So, if you do not have FT, CONSTANST is not able to activate the flowering. So, it meansthat there is an interaction, CONSTANST is upstream of FT and FT it is regulating throughthe FT. Similar kind of things if you look here another genetic interaction you can establishfrom your source. Again this is wild type if you have wild type,you have around 15 leaf at the time of flowering, but ft mutants it having delayed flowering.Another gene which is called SOC1, SOC1 is also delayed flowering, but if you combineFT and SOC1, the phenotype the plant is further showing further delayed flowering which meansthat this kind of phenotype is called additive phenotype. So, both the mutants are showingadditive phenotype. This tells that they are working in two independent pathways.So, FT is working through this pathway, SOC1 is working through this pathway. If you onlymutate FT, there is a effect. If you only mutate SOC1, there is a effect. But if youmutate both the parallel pathway the effect is enhanced.Similar kind of a genetic analysis has been done here to show that AGAMOUS-LIKE 17, anothergene which is even working through a different pathway, but this is also downstream of CONSTANST.As you can see in the constant mutant background, the expression of both FT as well AGL17 isdecreased. So, CONSTANST is activating AGL17 and AGL17 is also activating finally, AP1 and LFY. So, the final point of regulation is quiteconserved, but the upstream pathways are different quite different.So, this kind of genetic analysis will basically allow you to place all these mutants or allthese genes in a particular pathway and to generate a kind of genetic pathways. Anotherimportant thing is the genetic redundancy, genetic redundancy happens that sometime ifthere is a family of a genes, there are more than one members in a particular family, whathappens that they all or some of them are doing the same function, which means thatif you have mutant of one gene, you might not see a significant effect, but if you mutatemore than one genes you start seeing the effect this is called genetic redundancy.So, for example, if you look this is wild type plant and this is plethora. PLETHORAare AP2 domain containing transcription factor, very very important class of transcriptionfactor which regulates meristem function, stem cell maintenance as well as flowering,but here I am showing the effect in the root apical meristem.If you mutate plethora1, these are two different mutant of plethora1; you do not see a significantdefect in the root growth. Similarly, when you mutate plethora2 alone, you do not seea significant difference it is quite similar to the wild type, but when you make a doublemutant when you mutate both plethora1 as well as plethora2, you can see that root growthis very strongly inhibited. They are very very short rooted, which meansthat both plethora1 and plethora2, they are working in a genetically redundant mannerto regulate the root growth and single mutant of them, do not have a significant effect.Similar kind of things you can look here, here the effect is on the lateral root. So,primary root development is normal and these are ARF’s, ARF’s are Auxin Response Factorthey are also a class of transcription factor, but they works in the auxin mediated signallingpathway. So, for example, I will I will focus on two of auxin response factor this is auxarf19 this is arf7 and this is wild type. So, if you compare with the wild type, arf19and arf7 alone they do not have much effect on the lateral root development.So, you can see that primary roots are developed and these primary roots has significant numberof lateral roots, but when you combine both, when you make a double mutant where arf7 aswell as arf19 both are disrupted, you can see that primary root is very well grown itis equivalent to wild type, but this does not have any lateral roots. So, this alsosuggests that AUXIN RESPONSE FACTOR 7 and 19, they are genetically redundant in regulatinglateral root formation in Arabidopsis. So, this kind of genetic analysis will allow youto establish a genetic relationship between or among various regulators of a particulardevelopmental pathway.The next thing is that, if they are genetically interacting what would be the next question,are they physically interacting as well? If this is so, how to study them? There are manyway to study protein-protein interaction, some of them is highlighted here, this isyeast two hybrid, this is affinity purification based mass spectroscopy and this is bimolecularfluorescence complementation. These are few very commonly used. So, in yeast two hybrid,what we are doing? You take a gene and you basically. So, both the genes so here whatis important that you have to have both the gene cloned in a different vector one willhave to be fused with the bait and one with the prey.And if both the genes are basically interacting, they will interact and then the bait and preythey will come together and they will give some kind of signals. In affinity purificationmass spectroscopy, here basically based on the affinity column or you can have a kindof pull down and then you pull down some proteins and along with that proteins, there will belot of proteins are coming and then identify through the mass spectroscopy establish theiridentity to know that which genes are getting interacting. Similarly, this is in principlethis is similar to the yeast two hybrid, but here the mode of detection is the fluorescentbase. So, if you have a fluorescent protein whichhas two domains, but if these two domains are together then and only then they willgive the fluorescence. So, you clone them in the separately use one protein here, anotherprotein here, which you want to see whether they are interacting or not if these proteinsare interacting then they will bring both the domains together when a and b domainsare together they will give a kind of fluorescence signal then you can basically show whetherthey are interacting or not.So, here are a few cases of few examples of this protein-protein interaction. For example,if you look AP1 and SEPALLATA3 , AP3, PI you will study in the next class they are veryvery important genes which are regulating floral organ development in Arabidopsis andSEU S E U, this is another gene which is important and here what they have tried to check thatwhether SEU is interacting with these ABC genes or not. And, this has been done by theyeast two hybrid and what you can see this colour signal will tell there is a interaction,if there is no colour then there is no interaction. So, if you look this assay, you can clearlyfind that SEU is interacting only with AP1 and SEPALLATA3, but it is not interactingwith AP3, it is not directly or physically interacting with PI. Then if you have identifiedok AP1 and SEP3 are interacting with C1, you can also establish through yeast two hybridthat what is the domain of interaction. So, this AP1 and SEP3 both are MADS domain containingtranscription factor, they have one domain which is called MIK domain and another domainis C terminal domain. So, here specifically they have taken onlyMIK domains of both the factors and C terminal domains of both the factor and they have checkedthe interaction, you can clearly see that only C terminal domain are interaction showinginteraction. But, MIK domain is not showing interaction, which means that you can tellthat AP1 and SEPALLATA 3 they are interacting with SEU through their C terminal domain ok.Another assay this is done for another two very important transcription factor duringadventitious root development in rice. ERF3 is an AP2 domain containing transcriptionfactor, WOX11 is a homeobox domain containing transcription factor and both are very veryimportant for adventitious root development. And here through the pull down what they havedone. So, they have basically fused ERF3 with GST tag and WOX11 with His tag and you canpull down using anti GST antibody or anti His antibody and if you are using anti GSTfor pull down and then you can you can do immune blotting using anti His antibody.So, if ERF3 and WOX11 are interacting then if you pull down ERF3 WOX11 you can detectWOX11. If you pull down WOX11 you can detect ERF3 and this is in vitro way of doing thedetection and here you can see that, either you use anti GST or anti His in both the casesyou can detect the band. Another way of doing protein-protein interaction is Co-IP, co immuneprecipitation this you can do in vivo. So, you can basically make a transgenic plantwhere you already have a tagged protein. So, for example, this is FLAG tag. So, youcan tag your protein with any tag and then you can use antibody against the tag and youcan do the pull down from the total extract from the plant and then detect or do immuneblotting with the another gene which you want to show the interaction. This is from thesame study and this is your BiFC method and here you can see when these both the proteinsare interacting you will have yellow signal if they are not interacting there will notbe yellow signal.This is example of pull down followed by mass spectroscopy, I will not go in the detail,but why I want to tell here that you can do at the global level. So, you can tag a protein,you can use that tag in antibody against the tag, you can do the pull down and then youcan identify all the protein through mass spectroscopy at the global level to identifyall the proteins which are interacting with a particular protein.Then second study which is important is DNA-protein interaction as you know that most of the masterregulators of the developments are transcription factor. So, it is very important to identifywhat are their binding domain, what are the DNA elements where they bind and how theyregulates, what are their direct targets, what are their indirect targets. So, how todo there are again plenty of techniques available, but few of them I am discussing here.So, there are some in vitro way of doing, this is foot printing, electrophoretic mobilityshift assay and then yeast one hybrid in yeast, in yeast one hybrid you can use the DNA andthen identify what are the proteins which are identified to that DNA elements. Thesetwo are the more kind of physical assay to study the interaction FRET and SPR and thenChIP assay chromatin immunoprecipitation which is being very extensively used to study theprotein-protein interaction.So, this is one example of electrophoretic mobility shift assay, gel shift assay alsothis is called. So, in this case what you can do, you can take a putative binding siteDNA element you can make a small probe and then this probe can be labelled with radioactivenucleotide and then you mix with your protein and what happens that if your protein is bindingwith this elements and if you run the gel and detect the binding using any mutants youcan you will you can see a shift. So, now this is your free probe if your freeprobe is bound with the protein then you will detect a protein or at the higher molecularweight size, then you can tell that your protein is basically binding to the cis element. Toconfirm it, you can mutate your binding site putative binding site and show that when youare mutating this the binding is totally disappearing as you can see here there is no binding, buthere there is binding.Then ChIP assay as I say this is very important. So, chromatin immune precipitation this youcan do at the gene level, you can do at the global level the way you want. Here what youwant basically you have a putative binding sites. So, what you do, you allow your proteinto bind to the chromatin if you have a transcription factor it will go and bind to its target thenyou fix it you may cross link it. So, your protein will be permanently or covalentlybound to the DNA and then you extract total chromatin, make a small fragment and use antibodyagainst either your protein directly or if you have some tag associated with your proteinand pull down those chromatin region or those fragment of chromatins which are bound withyour protein and then you treat with proteinase. So, you will remove your protein now you havea DNA fragment only. So, either you take that DNA fragment andif you know what is the putative binding site you can design a specific primer and you canamplify through PCR, qRT PCR or qPCR or if you do not know the sequence you take thosefragments clone in some vectors sequence them and identify what are the binding sites. So,these are some examples for example, if you look these are the putative binding sitesof AGL24 which is MADS-domain transcription factor and this is tagged with the HA tag6HA tag. So, if what you can do, you can basicallydo the chromatin immunoprecipitation using antibody against the HA you will find thefragment and you want to see whether AGL24 is binding to SOC1 promoter or not these arethe putative binding sites for the AGL24 in the SOC1 promoter. And, then you check byRT PCR or by PCR simple genomic DNA PCR for the different region and what you can seethat, this region is showing maximum enrichment which means that this region is getting enrichedwhen you are doing chromatin immunoprecipitation for 24 which means that binding is very highat this region here this is low here this is low.But if you look this region the binding is very very low. Similar kind of study has beendone here, where a targets or direct targets of MADS1 another transcription factor in ricehas been studied. And here you can see, this analysis has been done to show that MADS1is directly associated with the chromatin of all these direct targets this is the wayto study protein-protein interaction or DNA-protein interaction and this is very important tobasically understand what is the mechanism of a transcription factor.Then you will move more to understand the regulatory interactions among the regulatorsand there the pathways which are being regulated by developmental regulators. One way of doingis that meta-analysis you can do the co-expression analysis and there are all these data whichare already published or submitted to the database all these database can be retrievedand lot of expression analysis and, actually there are lot of database has been generatedwhere you can you just put id of your gene, you can find what are the genes which areco-expressed with that gene. If they are co expressed with that gene, you may expect thatthey will be there must be some interaction they may there may be some interactions amongthemselves and this kind of study has been used to identify. For example, if you lookhere a global co expression network approach for connecting genes to specialized metabolicpathway in pathway. So, you if you combine data if you look theco expression then you can basically identify those genes which might be responsible orwhich might be functioning in a similar developmental pathway or similar pathway or similar metabolicpathway.Then another very important thing particularly with transcription factor is, if you wantto nail down that what is the mechanism how a particular transcription factor is regulatinga particular developmental pathway, you have to identify what are the genes which are regulatedby this transcription factor. And this you can do again as a gene levelor you can do at the global level. For identifying this kind of genes, the global techniqueslike microarray RNA sequencing can be used, if you quantify total expression of the genesor global expression of the genes in the wild type and mutant of a particular transcriptionfactor, you can identify what are the genes whose expression labels are altered in themutants and then you can establish that they are basically getting regulated.And then this kind of study has been taken very recently to identify global genes regulatedby PLETHORA genes. So, as I said there are multiple PLETHORA 1, 2, 3, 4, 5, 6, 7 andthen what it has been done. So, PLETHORA genes was fused with the glucocorticoidreceptor. If you recall one of the previous lecture there I have discussed what is glucocorticoidreceptor, this way you can control activity of your transcription factor by regulatingits translocation from cytoplasmic to nucleus and then when you induce and after induction,you can perform microarray and identify what are the genes which are getting induced orwhat are the genes which are getting repressed. So, by doing this they have basically identifiedthe genes which are shared by two PLETHORA three PLETHORA four PLETHORA five PLETHORAand all six PLETHORA and they have also identified what are the genes which are specificallyor uniquely regulated by either of the PLETHORA either activated or repressed. But, throughthis analysis through this global analysis for all these there was a very interestingobservation here that the all the activated genes if you look here.So, they are expressed in the meristematic genes. So, which means that in the meristematicgenes or in the meristem domains, the genes are mostly getting activated whereas, repressedgenes are mostly expressed in the elongation domain. So, by doing this kind of expressionor genetic network analysis you can predict genetic regulation.Similar kind of things has been done here, where architecture of gene regulatory networkscontrolling flower development in Arabidopsis thaliana. So, as I said that, the floweringstarts from the transition from the vegetative phase to reproductive phase and then oncethis transition has occurred, the floral primordia under are the floral meristem undergoes thedifferent developmental stage and targets of different developmental stage or transcriptionfactors at different developmental stage has been already identified in different studies.And, then if you can take all these data and if you can do the co-expression analysis,basically you can establish a regulatory relationship among the regulators and you can identifythe genes which are commonly regulated or the genes which are specifically regulatedby a particular regulators.So, this this will be probably last of today’s slide. So, here what I am trying to tell youthat if you want to identify, what is the direct downstream target of a transcriptionfactor, this could be one very good strategy which has been very successfully used withone transcription factor which is MADS1. So, if you remember my previous class, I havealready told that MADS1 is a very important regulator of floral organ development or flowerdevelopment in rice and if you mutate MADS1 basically, you will not have a proper flowerdevelopment and these plants are sterile.