BRD4 (HMV4275)

Bromodomain-containing protein 4 (BRD4) is a multifunctional protein coded by the BRD4 gene on chromosome 19p13.12. BRD4 is a member of the BET (bromodomain and extra terminal domain) family, which also includes BRD2, BRD3, and BRDT. As all other BET family members, BRD4 contains two bromodomains – termed BD1 and BD2 – that recognize acetylated lysine residues of histones or other proteins in a central hydrophobic pocket. BD1 mainly binds to histone acetylated lysine residues, whereas BD2 mainly binds to other non-histone acetylated lysine residues, such as acetylated lysine residues of RelA which is a component of NFkB. BRD4 acts as a passive scaffolding factor that recruits several chromatin and transcriptional regulatory factors. Because of its additional enzymatic functions (kinase and HAT activity) it also modifies and thereby regulates its interacting partners one of it is cMYC. In line with its multifunctional capacity, BRD4 plays a role in various biological processes including DNA replication, transcription, as well as cell cycle and signaling during stress responses. BRD4 dysfunction has been associated with a broad range of disease types including cancer, kidney, lung, heart, and inflammatory diseases, organ fibrosis, neuro-degenerative disorders, and auto immune diseases. BRD4 is an actively researched drug target, partly due to its critical regulatory role for cMYC. Multiple clinical trials are currently ongoing.

BRD4 is a nuclear protein which is ubiquitously expressed. Images describing the BRD4 staining pattern in normal tissues obtained by the antibody HMV4275 are shown in our “Normal Tissue Gallery”.

The findings described above are thus consistent with the the Human Protein Atlas (Tissue expression BRD4). BRD4 staining is seen almost in all normal cell types although the staining intensity is variable. Staining is absent (or only minimal) in syncytiotrophoblast nuclei of the mature placenta and in the most mature cells of the spermatogenesis in the testis.

Positive control = Kidney: A moderate to strong BRD4 staining should be seen in all cell types.

Negative control = Placenta (mature)*: BRD4 staining should be absent or only weak in nuclei of the syncytiotrophoblast but strong in other cell types.

Normal tissue gallery

Cancer tissue gallery

No data available at the moment

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein.

All images and data shown here and in our image galleries are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply HMV4275 at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

• The mechanisms involving BRD4 in disease are not fully understood.
• The utility of BRD4 measurement in cancer needs to be evaluated.
• The utility of BRD4 as a biomarker is not clear.
• BRD4 is an intensively investigated therapeutic target.

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

Orthogonal validation: For the antibody HMV4275 specificity is in line with data from three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, which are all compiled in the Human Protein Atlas (Tissue expression BRD4). In agreement with HMV4275 immunostaining data, high RNA expression occurs in all normal tissues. However, it must be understood that orthogonal validation is not optimal for assessing ubiquitously expressed protein.

Comparison of antibodies: True expression of BRD4 in cell types with documented BRD4 immunostaining by HMV4275 is validated by largely identical staining patterns obtained by a second, independent commercially available BRD4 antibody, termed “validation antibody” for virtually all normal tissues. In particular, the use of the validation antibody confirmed characteristic variabilities of BRD4 staining in several tissue types. These included the absence or a significant reduction of BRD4 staining in syncytiotrophoblast nuclei of the mature placenta, and distinctive differences in the staining intensity between different cell types in non-keratinizing squamous epithelium, gastric mucosa, duodenum, colorectal mucosa, liver, salivary glands, prostate, testis, and in the grey matter of the cerebrum. That cerebral neurons were found BRD4 positive by the validation antibody and not by HMV4275 was interpreted as an antibody specific cross-reactivity of the validation antibody while a cytoplasmic staining of heart and skeletal muscle cells which was only seen by HMV4275 was considered an antibody specific cross-reactivity of HMV4275.

Normal tissue gallery